bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Analysis of
Mycobacterium kansasii
Isolates from the United States
Authors: Zhang Yansheng , Mann Linda B. , Wilson Rebecca W. , Brown-Elliott Barbara A. , Vincent Véronique , Iinuma Yoshitsugu , Wallace Richard J.
Published: January 2004

We studied the population genetics of
Mycobacterium kansasii
isolates from the United States by PCR restriction enzyme analysis (PRA) of the 441-bp Telenti fragment of the
hsp
-65 gene and pulsed-field gel electrophoresis (PFGE) of genomic DNA with the restriction endonucleases
Ase
I,
Dra
I, and
Xba
I, and we compared the patterns to those previously reported from France and Japan. By PRA, 78 of 81 clinical isolates (96%) from the United States belonged to subspecies I. With PFGE, 28
Ase
I patterns, 32
Dra
I patterns, and 35
Xba
I patterns were produced. PFGE showed marked clonality of the U.S. isolates, with differences between genotypes involving only one or two bands. Isolates within Texas showed lower pattern diversity than those from different states. With
Dra
I, 31 of 71 isolates (44%) had the same common PFGE pattern, which matched the predominant pattern in France (pattern Ia), determined by Picardeau et al. (M. Picardeau, G. Prod'hom, L. Raskine, M. P. LePennec, and V. Vincent, J. Clin. Microbiol.
35:
25-32, 1997), and in Japan (type M), determined by Iinuma et al. (Y. Iinuma, S. Ichiyama, Y. Hasegawa, K. Shimokata, S. Kawahara, and T. Matsushima, J. Clin. Microbiol.
35:
596-599, 1997). With
Ase
I, 42% of isolates produced a common pattern indistinguishable from the common pattern seen in French isolates (Ia) and with only one band difference from the common pattern (type M) in Japan. This study demonstrates that subspecies I is the predominant subspecies of
M. kansasii
among clinical isolates in the United States, as it is in Europe and Japan, and that genotype I is highly clonal worldwide, with the same major genotype responsible for human infection. The fact that a single clone of
M. kansasii
is responsible for most cases of human disease suggests that specific virulence factors may be associated with this specific genotype.
Utility of the Focus Technologies West Nile Virus Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Testing Cerebrospinal Fluid
Authors: Prince Harry E. , Lape'-Nixon Mary , Moore Ronald J. , Hogrefe Wayne R.
Published: January 2004

Focus Technologies has developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) kit that utilizes recombinant West Nile virus (WNV) antigens to detect WNV IgM in serum. We evaluate here the utility of the kit for detecting WNV IgM in cerebrospinal fluid (CSF). The sensitivity was evaluated by using 52 CSF specimens from the 2002 WNV season that were positive in both the Public Health Service Laboratories WNV IgM ELISA and an in-house WNV IgM ELISA with native WNV antigen. The specificity was evaluated with two groups of specimens: (i) 73 CSF specimens submitted for in-house WNV IgM ELISA testing from February through April 2003 and yielding a negative WNV IgM result and (ii) 60 CSF specimens determined to be positive for another virus by PCR testing. Using these 185 CSF specimens at a screening dilution of 1:2, the kit was determined to be 100% sensitive and 100% specific. Endpoint titers were determined for 20 IgM-positive CSF specimens by testing serial twofold dilutions and ranged from 1:8 to 1:512. Index values (specimen absorbance value/calibrator absorbance value) for the screening dilution (1:2) showed no correlation with IgM titers, whereas index values for higher dilutions showed significant correlation with IgM titers. CSF screening dilutions of greater than 1:2 are not recommended, however, due to the risk of obtaining false-negative results. These findings show that the Focus Technologies WNV IgM capture ELISA, when utilized as recommended, offers accurate qualitative detection of WNV IgM in CSF specimens.
Human Metapneumovirus Infection in Japanese Children
Authors: Ebihara Takashi , Endo Rika , Kikuta Hideaki , Ishiguro Nobuhisa , Ishiko Hiroaki , Hara Michimaru , Takahashi Yutaka , Kobayashi Kunihiko
Published: January 2004

Human metapneumovirus (hMPV) has been recently discovered as an etiological agent of acute respiratory infections. Our purpose was to asses the virological and clinical features of children with respiratory infections caused by hMPV. We examined 658 nasopharyngeal swab samples obtained from 637 children with respiratory infections for hMPV by using reverse transcription-PCR (RT-PCR). A total of 268 samples from 637 children were inoculated onto tertiary monkey kidney cells. A total of 36 serum samples (26 in the acute phase and 10 in the convalescent phase) from the 26 hMPV-positive children were tested for immunoglobulin G (IgG) and IgM antibodies to hMPV by using an indirect immunofluorescence assay. We detected hMPV in 57 (8.9%) of the 637 samples by using RT-PCR and isolated 7 (2.6%) hMPV strains of the 268 samples in cell cultures. A total of 12 (46.2%) of 26 hMPV-positive children were suspected to have primary infection with hMPV as determined by an indirect immunofluorescence assay. The infected children were diagnosed as having wheezy bronchitis (36.8%), upper respiratory tract infection (26.3%), bronchitis (22.8%), and pneumonia (14.0%). We showed that two hMPV groups were circulating in different regions during the same period and that reinfection with hMPV frequently occurs in childhood. The RT-PCR test is the most sensitive test for detection of hMPV, and a serological test may be useful to differentiate between primary infection and reinfection with hMPV.
Typical Enteroaggregative
Escherichia coli
Is the Most Prevalent Pathotype among
E. coli
Strains Causing Diarrhea in Mongolian Children
Authors: Sarantuya Jav , Nishi Junichiro , Wakimoto Naoko , Erdene Shirchin , Nataro James P. , Sheikh Jalaluddin , Iwashita Mayumi , Manago Kunihiro , Tokuda Koichi , Yoshinaga Masao , Miyata Koichiro , Kawano Yoshifumi
Published: January 2004

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic
Escherichia coli
. In Mongolia, the epidemiology of diarrheagenic
E. coli
has not been well studied. A total of 238
E. coli
strains from children with sporadic diarrhea and 278
E. coli
strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic
E. coli
(EPEC)
eae
,
tir
, and
bfpA
; enterotoxigenic
E. coli
(ETEC)
lt
and
st
; enteroinvasive
E. coli
(EIEC)
ipaH
; enterohemorragic
E. coli stx1
and
stx2
; and enteroaggregative
E. coli
(EAEC)
aggR
and
astA
. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%;
P
= 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes—
aggA
, 2 (5.5%);
aafA
, 4 (11.1%);
agg-3a
, 5 (13.8%);
aap
, 8 (22.2%);
aatA
, 11 (30.5%);
capU
, 9 (25.0%);
pet
, 6 (16.6%); and
set
, 3 (8.3%)—and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains.
Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification
Authors: Ihira Masaru , Yoshikawa Tetsushi , Enomoto Yoshihiko , Akimoto Shiho , Ohashi Masahiro , Suga Sadao , Nishimura Naoko , Ozaki Takao , Nishiyama Yukihiro , Notomi Tsugunori , Ohta Yoshinori , Asano Yoshizo
Published: January 2004

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.
Comparison of DNA Dot Blot Hybridization and Lancefield Capillary Precipitin Methods for Group B Streptococcal Capsular Typing
Authors: Borchardt Stephanie M. , Foxman Betsy , Chaffin Donald O. , Rubens Craig E. , Tallman Patricia A. , Manning Shannon D. , Baker Carol J. , Marrs Carl F.
Published: January 2004

Group B streptococci (GBS) (
Streptococcus agalactiae
) are a major cause of sepsis and meningitis in neonates and infants and of invasive disease in pregnant women, nonpregnant, presumably immunocompromised adults, and the elderly. Nine GBS serotypes based on capsular polysaccharide antigens have been described. The serotype distributions among invasive and colonizing isolates differ between pediatric and adult populations and have changed over time. Thus, periodic monitoring of GBS serotype distributions is necessary to ensure the proper formulation and application of an appropriate GBS vaccine for human use and to detect the emergence of novel serotypes. Since the mid-1990s, the proportion of GBS isolates that are nontypeable by standard serologic methods has increased, creating a need for more sensitive typing methods. We describe a typing method that uses DNA dot blot hybridization with probes generated by PCR from the GBS capsular genes for serotypes Ia, Ib, and II to VIII. PCR primers were designed to amplify type-specific GBS capsular gene sequences. Gene probes were constructed from the PCR products and used to classify isolates based on hybridization profiles. A total of 306 previously serotyped invasive and colonizing isolates were used to compare our dot blot capsular typing (DBCT) identification method with Lancefield serotyping (LS). A dot blot capsular type was assigned to 99% (303 of 306) of the isolates, whereas 273 of 306 isolates (89%) were assigned a Lancefield serotype. The overall agreement between the methods was 95% (256 of 270 isolates typeable by both methods). We conclude that the DBCT method is a specific and useful alternative to the commonly used LS method.
Prevalence and Genetic Diversity of Human Astroviruses in Mexican Children with Symptomatic and Asymptomatic Infections
Authors: Méndez-Toss Martha , Griffin Dixie D. , Calva Juan , Contreras Juan F. , Puerto Fernando I. , Mota Felipe , Guiscafré Héctor , Cedillo Roberto , Muñoz Onofre , Herrera Ismael , López Susana , Arias Carlos F.
Published: January 2004

The prevalence and type diversity of human astroviruses (HAstV) in children with symptomatic and asymptomatic infections were determined in five localities of Mexico. HAstV were detected in 4.6 (24 of 522) and 2.6% (11 of 428) of children with and without diarrhea, respectively. Genotyping of the detected strains showed that at least seven (types 1 to 4 and 6 to 8) of the eight known HAstV types circulated in Mexico between October 1994 and March 1995. HAstV types 1 and 3 were the most prevalent in children with diarrhea, although they were not found in all localities studied. HAstV type 8 was found in Mexico City, Monterrey, and Mérida; in the last it was as prevalent (40%) as type 1 viruses, indicating that this astrovirus type is more common than previously recognized. A correlation between the HAstV infecting type and the presence or absence of diarrheic symptoms was not observed. Enteric adenoviruses were also studied, and they were found to be present in 2.3 (12 of 522) and 1.4% (6 of 428) of symptomatic and asymptomatic children, respectively.
Hepatitis C Genotyping by Denaturing High-Performance Liquid Chromatography
Authors: Liew Michael , Erali Maria , Page Sam , Hillyard David , Wittwer Carl
Published: January 2004

Determination of the hepatitis C virus (HCV) genotype for infected patients increasingly has become accepted as the standard of care. Genotype assignment helps in assessing disease prognosis and assists in establishing the appropriate duration of treatment. The great genetic diversity of HCV, with 11 major genotypes and >70 subtypes, contributes to the technical difficulty of genotype testing. While the “gold standard” for testing is nucleic acid sequencing, a variety of hybridization assays, including the line probe assay, have been developed to provide more rapid and accessible forms of testing. The aim of this study was to determine whether denaturing high-performance liquid chromatography (dHPLC) could be used as a clinical method for distinguishing HCV genotypes 1, 2, 3, and 4. A portion of the 5′ untranslated region of the HCV genome was amplified by heminested multiplex reverse transcription PCR. The two amplicons then were analyzed by dHPLC analysis and compared to the genotypes determined by sequence analysis. After 115 specimens were analyzed as standards, 200 masked specimens (specimens whose identity was not known before testing) were analyzed to determine the concordance of the assay. The assay had a concordance of 96% at the genotype level and a concordance of 87% at the subtype level. However, the dHPLC method was not as accurate as other reported methods of HCV genotyping. This is the first time that HCV genotyping has been performed by dHPLC.
Simple DNA Extraction Method for Dried Blood Spots and Comparison of Two PCR Assays for Diagnosis of Vertical Human Immunodeficiency Virus Type 1 Transmission in Rwanda
Authors: Fischer A. , Lejczak C. , Lambert C. , Servais J. , Makombe N. , Rusine J. , Staub T. , Hemmer R. , Schneider F. , Schmit J. C. , Arendt V.
Published: January 2004

Dried blood spots (DBS) on filter paper facilitate the collection, transport, and storage of blood samples for laboratory use. A rapid and simple DNA extraction procedure from DBS was developed and evaluated for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in children by an in-house nested-PCR assay on three genome regions and by the Amplicor HIV-1 DNA prototype assay version 1.5 (Roche Molecular Systems). A total of 150 samples from children born to HIV-1-infected mothers were collected in Kigali, Rwanda, in parallel as DBS and as peripheral blood mononuclear cell (PBMC) pellets. The results obtained on DBS by the two PCR assays were compared to the results of nested PCR on PBMCs. Of 150 PBMC samples, 10 were positive, 117 were negative, and 23 were indeterminate for HIV-1 infection. In DNA extracted from filter papers and amplified by using the in-house nested PCR, 9 of these 10 positive samples (90%) were found to be positive, and 1 was found to be indeterminate (only the
pol
region could be amplified). All of the negative samples and all of the 23 indeterminate samples tested negative for HIV-1 infection. When we used the Amplicor DNA test on DBS, all of the 10 PBMC-positive samples were found to be positive and all of the 23 indeterminate samples were found to be negative. Of the PBMC-negative samples, 115 were found to be negative and 2 were found to be indeterminate. We conclude that this simple rapid DNA extraction method on DBS in combination with both detection methods gave a reliable molecular diagnosis of HIV-1 infection in children born to HIV-infected mothers.
Increased Sensitivity of Mannanemia Detection Tests by Joint Detection of α- and β-Linked Oligomannosides during Experimental and Human Systemic Candidiasis
Authors: Sendid Boualem , Jouault Thierry , Coudriau Richard , Camus Daniel , Odds Frank , Tabouret Marc , Poulain Daniel
Published: January 2004

An enzyme immunoassay (EIA)—the commercially available Platelia
Candida
antigen test—developed for the diagnosis of systemic candidiasis is based on the detection of α-linked oligomannose residues (α-Man) released from
Candida
cells into the serum. This test has good specificity but has to be repeated frequently because of the rapid clearance of detectable mannanemia. We have developed a second EIA based on detection of beta-linked oligomannoses (β-Man), since β-Man are linked to different
Candida
molecules and interact differently with the host immune system and endogenous lectins and should therefore present different kinetics of serum clearance. In a guinea pig model of
Candida albicans
systemic infection, the relative amounts of detectable α- and β-Man differed considerably according to the virulence of the strain, the infecting dose, and the time after challenge that serum samples were drawn. Detection of α-Man was more sensitive per serum sample than that of β-Man, and the sensitivity for the combination reached 90%. The same tests were applied to 90 sera from 26 patients selected retrospectively for having been infected with the most-pathogenic
Candida
species:
C. albicans
(19),
C. tropicalis
(4), and
C. glabrata
(3). A total of 22 patients had positive antigenemia, 4 had α-mannanemia, 4 had β-mannanemia, and 14 showed the presence of both. For the patients showing the presence of both forms of mannanemia, the use of both tests enhanced the duration of the detection of mannanemia. Mannanemia was correlated with early clinical symptoms and isolation of
Candida
in culture, which occurred in 55% of the patients at an average of 4.7 days after the first positive mannanemia test result. A combination of the two tests had a cumulated specificity of 95%, and positive and negative predictive values were 79 and 97%, respectively. These findings provide evidence for different kinetics of β- and α-Man circulation during experimental and human candidiasis and suggest the joint detection of both types of epitopes as a rational approach contributing to increases in the sensitivity and earliness of diagnosis.
Decision Support Tools for Clinical Diagnosis of Disease in Cows with Suspected Bovine Spongiform Encephalopathy
Authors: Saegerman C. , Speybroeck N. , Roels S. , Vanopdenbosch E. , Thiry E. , Berkvens D.
Published: January 2004

Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.
Detection of the
Bacillus anthracis gyrA
Gene by Using a Minor Groove Binder Probe
Authors: Hurtle William , Bode Elizabeth , Kulesh David A. , Kaplan Rebecca Susan , Garrison Jeff , Bridge Deanna , House Michelle , Frye Melissa S. , Loveless Bonnie , Norwood David
Published: January 2004

Identification of chromosomal markers for rapid detection of
Bacillus anthracis
is difficult because significant chromosomal homology exists among
B. anthracis
,
Bacillus cereus
, and
Bacillus thuringiensis
. We evaluated the bacterial
gyrA
gene as a potential chromosomal marker for
B. anthracis
. A real-time PCR assay was developed for the detection of
B. anthracis
. After analysis of the unique nucleotide sequence of the
B. anthracis gyrA
gene, a fluorescent 3′ minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102
Bacillus
strains. The assay was found to be specific for all 43 strains of
B. anthracis
tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the
gyrA
gene as a specific chromosomal marker for
B. anthracis
.
Characterization of Group C and G Streptococcal Strains That Cause Streptococcal Toxic Shock Syndrome
Authors: Hashikawa Shinnosuke , Iinuma Yoshitsugu , Furushita Manabu , Ohkura Teruko , Nada Toshi , Torii Keizo , Hasegawa Tadao , Ohta Michio
Published: January 2004

Twelve strains (the largest number ever reported) of group C and G
1
streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as
Streptococcus dysgalactiae
subsp.
equisimilis
, and one strain was identified as
Streptococcus equi
subsp.
zooepidemicus
. We found that it was the first reported case of STSS caused by
S. equi
subsp.
zooepidemicus
. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the
S. dysgalactiae
strains belonged to clusters I and II, both of which were closely related. The
emm
types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular
S. dysgalactiae
strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the
spegg
gene was detected in seven
S. dysgalactiae
strains, with none of the other superantigen genes being detected in any of the strains. However, the
sagA
gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.
Development of a Dipstick Assay for Detection of
Leishmania
-Specific Canine Antibodies
Authors: Schallig Henk D. F. H. , Cardoso Luís , Hommers Marieke , Kroon Nel , Belling Guus , Rodrigues Manuela , Semião-Santos Saul J. , Vetter Hans
Published: January 2004

A dipstick assay, based on
Leishmania infantum
antigen, for the rapid detection of
Leishmania
-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ value, 0.86), between the dipstick test and FAST (91.8%; κ value, 0.82), and between the DAT and FAST (95.2%; κ value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys.
Demonstration of
Streptococcus mutans
with a Cell Wall Polysaccharide Specific to a New Serotype,
k
, in the Human Oral Cavity
Authors: Nakano Kazuhiko , Nomura Ryota , Nakagawa Ichiro , Hamada Shigeyuki , Ooshima Takashi
Published: January 2004

Streptococcus mutans
organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of
S. mutans
. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2,500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-
c
, -
e
, and -
f
antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference
S. mutans
strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype
k
. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the
gluA
gene of strain MT8148, which showed biological properties similar to those of serotype
k
of
S. mutans
. Serotype
k
oral isolates were less susceptible to phagocytosis, as were the
gluA
-inactivated mutant of strain MT8148 and blood isolates. These results indicate that
S. mutans
serotype
k
strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.
Diagnostic Assessment of
Mycoplasma genitalium
in Culture-Positive Women
Authors: Baseman Joel B. , Cagle Marianna , Korte Jeffrey E. , Herrera Caleb , Rasmussen Wanda G. , Baseman Janet G. , Shain Rochelle , Piper Jeanna M.
Published: January 2004

Detection of
Mycoplasma genitalium
-mediated, chlamydia-negative nongonococcal urethritis and other
M. genitalium
-linked infectious etiologies has been very challenging. Although
M. genitalium
is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of
M. genitalium
in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in
M. genitalium
culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of
M. genitalium
-host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable
M. genitalium
organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to
M. genitalium
based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.
Multicenter Evaluation of a New, Automated Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus-Specific Antibodies and Antigen
Authors: Sickinger Eva , Stieler Myriam , Kaufman Boris , Kapprell Hans-Peter , West Daniel , Sandridge Arnold , Devare Sushil , Schochetman Gerald , Hunt J. C. , Daghfal David
Published: January 2004

A collaborative multicenter study was conducted to evaluate the sensitivity, specificity, and precision of a three-step, fully automated, qualitative microparticle-based enzyme-linked immunoassay (AxSYM HIV Ag/Ab Combo; Abbott Laboratories), designed to simultaneously detect (i) antibodies against human immunodeficiency virus type 1 (HIV-1) and/or type 2 (HIV-2) and (ii) HIV p24 antigen. A significant reduction in the HIV seroconversion window was achieved by combining detection of HIV antibodies and antigen into a single assay format. For 22 selected, commercial HIV seroconversion panels, the mean time of detection with the combined-format HIV antigen-antibody assay was reduced by 6.15 days compared to that with a similar third-generation single-format HIV antibody assay. The quantitative sensitivity of the combination assay for the p24 antigen (17.5 pg/ml by use of the p24 quantitative panel VIH SFTS96′) was nearly equivalent to that of single-format antigen tests. The combination assay demonstrated sensitive (100%) detection of anti-HIV immunoglobulin in specimens from individuals in CDC stages A, B, and C and from individuals infected with different HIV-1 group M subtypes, group O, or HIV-2. The apparent specificity for hospitalized patients (
n
= 1,938) was 99.90%. In a random population of 7,900 volunteer blood donors, the specificity (99.87%) was comparable to that of a third-generation single-format HIV antibody assay (99.92%) on the same donor specimens. In addition, the combination assay was robust to potential interfering specimens. The precision of the combination was high, with intra- and interrun variances of ≤9.3% for each precision panel specimen or assay control and ≤5.3% for the negative assay control.
IS
6110
Mediates Increased Transcription of the
phoP
Virulence Gene in a Multidrug-Resistant Clinical Isolate Responsible for Tuberculosis Outbreaks
Authors: Soto Carlos Y. , Menéndez M. Carmen , Pérez Esther , Samper Sofía , Gómez Ana B. , García María J. , Martín Carlos
Published: January 2004

Drug resistance in
Mycobacterium tuberculosis
complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR)
M. tuberculosis
complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of
M. bovis
. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS
6110
. Here, we mapped and sequenced the regions flanking the two copies of IS
6110
in the B strain. Ligation-mediated PCR showed that one of these IS
6110
copies is located within the promoter region of
phoP
, a transcriptional regulator that is essential for
M. tuberculosis
virulence. We used PCR to screen 219 MDR
M. tuberculosis
complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS
6110
in the
phoP
promoter. To determine whether IS
6110
affects
phoP
promoter activity in the B strain, we individually cloned the
phoP
gene and its promoter region (including IS
6110
from the B strain and the equivalent region from
M. tuberculosis
without IS
6110
as a control) into a mycobacterial replicative plasmid and transformed
M. smegmatis
with the resulting plasmid. Primer extension analysis showed that
phoP
transcription was strongly upregulated when the promoter region contained IS
6110
, as in the case of the B strain.
Typing of Clinical and EnvironmentalAeromonassp. Strains by Random Amplified Polymorphic DNA PCR, Repetitive Extragenic Palindromic PCR, and Enterobacterial Repetitive Intergenic Consensus Sequence PCR
Authors: Szczuka Ewa , Kaznowski Adam
Published: January 2004

A collection of 120 strains isolated from stool specimens collected from humans suffering from gastroenteritis and from environmental samples were analyzed by random amplified polymorphic DNA PCR (RAPD), repetitive extragenic palindromic PCR (REP-PCR), and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). Species ofAeromonoas hydrophila,A. bestiarum,A. salmonicida,A. caviae,A. media, andA. veroniirevealed clonal structure. There was no dominant clone causing gastroenteritis in humans. Moreover, there was no genetic similarity between clinical and environmental strains ofAeromonassp. isolated from different geographical areas as well as from the same geographical area. Some clones colonized specific ecosystems, e.g., drinking water distribution systems. RAPD and ERIC-PCR methods had the same discriminatory power and proved to be useful for epidemiological investigation and population genetic analysis ofAeromonasspp., whereas REP-PCR was less effective for differentiating the isolates ofAeromonasspp.
Fluorescent Amplified Fragment Length Polymorphism Genotyping of
Campylobacter jejuni
and
Campylobacter coli
Strains and Its Relationship with Host Specificity, Serotyping, and Phage Typing
Authors: Hopkins Katie L. , Desai Meeta , Frost Jennifer A. , Stanley John , Logan Julie M. J.
Published: January 2004

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to 276
Campylobacter jejuni
strains and 87
Campylobacter coli
strains isolated from humans, pigs, cattle, poultry, and retail meats to investigate whether certain FAFLP genotypes of
C. jejuni
and
C. coli
are associated with a particular host and to determine the degree of association between FAFLP-defined genotypes and heat-stable serotypes and/or phage types. Within
C. coli
, the poultry strains clustered separately from those of porcine origin. In contrast, no evidence of host specificity was detected among
C. jejuni
strains. While
C. coli
strains show host specificity by FAFLP genotyping, C
. jejuni
strains that are genotypically similar appear to colonize a range of hosts, rather than being host adapted. Some serotypes and/or phage types (
C. jejuni
serotype HS18, phage type PT6, and serophage type HS19/PT2 and
C. coli
HS66, PT2, and HS56/PT2) were the most homogeneous by FAFLP genotyping, while others were more heterogeneous (
C. jejuni
HS5 and PT39, and
C. coli
HS24 and PT44) and therefore poor indicators of genetic relatedness between strains. The lack of host specificity in
C. jejuni
suggests that tracing the source of infection during epidemiological investigations will continue to be difficult. The lack of congruence between some serotypes and/or phage types and FAFLP genotype underlines the need for phenotypic testing to be supplemented by genotyping. This study also demonstrates how, in general, FAFLP generates “anonymous” genetic markers for strain characterization and epidemiological investigation of
Campylobacter
in the food chain.
Nonvalue of Culturing Cerebrospinal Fluid for Fungi
Authors: Barenfanger Joan , Lawhorn Jerry , Drake Cheryl
Published: January 2004

No studies have evaluated the efficacy of culturing cerebrospinal fluid (CSF) for fungi. Because of the facts that the most common fungi responsible for meningitis grow well in media utilized for routine bacterial cultures and that cryptococcal antigen tests are commonly ordered, the efficacy of routinely performing fungal cultures specifically to recover fungi has been questioned. We examined data from 1,225 samples of CSF which were cultured for both bacteria and fungi. Twelve specimens yielded fungi, 10 from fungal cultures and 8 from bacterial cultures.
Cryptococcus neoformans
was found in 10 specimens,
Candida albicans
was found in 1, and a
Cladosporium
sp. was found in 1. Eight of 12 positive specimens had concordant culture results. The discordant cases were one specimen that was bacterial culture positive but fungal culture negative and three specimens that were fungal culture positive but bacterial culture negative. Of the latter discrepant cultures, one had fungal contamination only and the other two were positive for cryptococcal antigen. Therefore, omitting the fungal cultures on these specimens would not adversely impact patients. When both cryptococcal antigen tests and bacterial cultures are ordered routinely, eliminating fungal cultures on CSF would have had no impact on the patients in this study. All the clinically significant fungi were detected by the cryptococcal antigen test and/or bacterial culture. With a few exceptions, the combined use of cryptococcal antigen test and bacterial cultures of CSF could replace routine fungal cultures of CSF. Exceptions include settings where fungal pathogens other than
Cryptococcus
and
Candida
remain important causes of meningitis.
Validation of Roche COBAS Amplicor Assay for Detection of
Chlamydia trachomatis
in Rectal and Pharyngeal Specimens by an
omp1
PCR Assay
Authors: Lister N. A. , Tabrizi S. N. , Fairley C. K. , Garland S.
Published: January 2004

Screening guidelines for men who have sex with men (MSM) recommend testing of extragenital sites (pharyngeal and rectal) for gonorrhoea and chlamydia. Testing of specimens from these sites is not validated by most commercial nucleic amplification tests, such as the COBAS Amplicor assay. To investigate the utility of the COBAS Amplicor assay for detection of
Chlamydia trachomatis
in extragenital specimens, this study developed and evaluated confirmatory tests using the
omp1
gene as an alternative target for amplification by PCR. Of anal and throat swabs collected from men in male-only saunas, 52 swabs that tested
C. trachomatis
positive by COBAS Amplicor and 30 swabs that tested as negative were included for confirmatory
omp1
PCR testing. A total of 49 (94%) COBAS Amplicor-positive samples were confirmed by the
omp1
PCR. A substantial proportion of specimens were confirmed by using a nested
omp1
PCR (27%). Not confirmed by any
omp1
PCR were three anal swabs (6%). It is most probable that these samples contained lower bacterial levels that were near or below the detection level of the
omp1
PCR assays. The findings of this study support the confident reporting of
C. trachomatis
detected by COBAS Amplicor in extragenital specimens and support the utility of this assay as a screening test for MSM.
Surveillance Network for Herpes Simplex Virus Resistance to Antiviral Drugs: 3-Year Follow-Up
Authors: Danve-Szatanek C. , Aymard M. , Thouvenot D. , Morfin F. , Agius G. , Bertin I. , Billaudel S. , Chanzy B. , Coste-Burel M. , Finkielsztejn L. , Fleury H. , Hadou T. , Henquell C. , Lafeuille H. , Lafon M. E. , Le Faou A. , Legrand M. C. , Maille L. , Mengelle C. , Morand P. , Morinet F. , Nicand E. , Omar S. , Picard B. , Pozzetto B. , Puel J. , Raoult D. , Scieux C. , Segondy M. , Seigneurin J. M. , Teyssou R. , Zandotti C.
Published: January 2004

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains . We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3,900 isolated strains among 3,357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.
Serotype 14 Variants of the France 9V
−3
Clone from Baltimore, Maryland, Can Be Differentiated by the
cpsB
Gene
Authors: McEllistrem M. Catherine , Noller Anna C. , Visweswaran Shyam , Adams Jennifer M. , Harrison Lee H.
Published: January 2004

European serotype 14 variants of the France 9V
−3
clone, which have arisen through recombination events involving the penicillin binding protein 1a (
pbp1a
) gene, have
cpsB
sequences distinct from those of the 9V
−3
clone. Serotype 14 variants of the 9V
−3
clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible
Streptococcus pneumoniae
strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST),
pbp1a
PCR restriction profiles, and
cpsB
and
pbp1a
sequences. The
cpsB
genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V
−3
clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V
−3
clone nor to the R6 strain from positions 1498 to 1710 of the
pbp1a
gene. All serotype 14 strains had
cpsB
sequences that differed by ≤1% (0 to 5 of 476 bp) from each other and that were ≥16% (78 to 83 of 476 bp) divergent from that of the 9V
−3
clone. Allowing for a 2-bp difference in the
cpsB
sequence resulted in the highest correlation between the
cpsB
gene and serotype. Overall, 95% (84 of 88) of the strains were classified correctly by serotype with the
cpsB
sequence. The distal recombination site of the Baltimore serotype 14 variants of the 9V
−3
clone was not identical to that of the European serotype 14 variants. The
cpsB
gene was serotype specific regardless of whether capsular switching occurred. Although the correlation between serotype and the
cpsB
sequence was high, the overall diversity of the
cpsB
gene within a serotype likely will limit the role of this gene in a sequence-based serotyping method.
Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus
Authors: Parida Manmohan , Posadas Guillermo , Inoue Shingo , Hasebe Futoshi , Morita Kouichi
Published: January 2004

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and
Bst
DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 10
4
PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the
Flavivirus
group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.
Prevalence of
Bartonella henselae
in Italian Stray Cats: Evaluation of Serology To Assess the Risk of Transmission of
Bartonella
to Humans
Authors: Fabbi Massimo , De Giuli Luciana , Tranquillo Massimo , Bragoni Roldano , Casiraghi Maurizio , Genchi Claudio
Published: January 2004

Bartonella henselae
is the major etiological agent of cat scratch disease in humans. Cats act as the natural reservoir of
B. henselae
and can transmit the infection to humans by a bite or scratch. The prevalence of
B. henselae
in cat populations was evaluated by serological and bacteriological tests. A total of 769 stray cats from three urban and three rural areas in northern Italy were sampled between January 1999 and December 2000. The positive and the negative predictive values of serological tests with respect to bacteremic status were evaluated. Tests of a total of 140 cats (18%) resulted in detection of bacteremia. A total of 540 cats were tested by serology; 207 (38%) were seropositive. Of the 531 cats tested by both methods, the results for 65 (12.2%) showed both bacteremia detection and seropositivity. The molecular typing of the isolates showed that 20.6% of bacteremic cats were infected with
B. henselae
type I strain, 61.1% were infected with
B. henselae
type II, and 18.3% were coinfected with both. A statistically significant difference in antibody and bacteremia prevalences among geographical areas was detected. Statistical analysis showed no association between characteristics such as seroprevalence-bacteremic status, sex, general health status, and the presence of ectoparasites. The negative predictive value of serological test was 84.7%, and the positive predictive value was 31.8%. Receiving operator characteristic analysis of the data showed that serological tests had a low predictive value in relation to the bacteremic status of a cat; in surveys aimed at assessing the real risk of
B. henselae
infection in a human population, therefore, we suggest the use of blood culture as the reference test. Nevertheless, both blood culture assays and serological tests for
Bartonella
infection should be performed for a complete evaluation of the health status of cats.