bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific Detection of
Fusarium
Species in Blood and Tissues by a PCR Technique
Authors: Hue Francois-Xavier , Huerre Michel , Rouffault Marie Ange , de Bievre Claude
Published: December 2019

Fusarium
species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA of
Fusarium
species. The DNAs of the main
Fusarium
species and
Neocosmospora vasinfecta
but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of
Fusarium solani
DNA by ethidium bromide staining. To test the ability of this PCR system to detect
Fusarium
DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected
Fusarium
DNA in mouse tissues and in spiked human blood. Furthermore,
F. solani
,
Fusarium moniliforme
, and
Fusarium oxysporum
were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or several
Fusarium
species. The method developed may be useful for the rapid detection and identification of
Fusarium
species both from culture and from clinical samples.
Abiotrophia elegans
Strains Comprise 8% of the Nutritionally Variant Streptococci Isolated from the Human Mouth
Authors: Sato Setsuko , Kanamoto Taisei , Inoue Masakazu
Published: December 2019

Ninety-one isolates of nutritionally variant streptococci (NVS) that were previously isolated from the human mouth were regarded as consisting of 7
Streptococcus defectivus
isolates, 78
Streptococcus adjacens
isolates, and 6
Gemella morbillorum
isolates. However, recent references to the taxonomic reclassification of NVS, from
S. defectivus
to
Abiotrophia defectiva
and from
S. adjacens
to
Abiotrophia adiacens
, and the newly introduced species
Abiotrophia elegans
as a third
Abiotrophia
species, emphasize the need for genetic analyses for identification of NVS. When PCR-restriction fragment length polymorphism (RFLP) and phylogenetic distances were examined based on 16S rRNA gene sequences, the results indicated that 7 of the 91 NVS isolates were closely related to
A. elegans
. These seven isolates consisted of four isolates previously identified as
G. morbillorum
and three isolates previously identified as
S. adjacens
. Two isolates previously identified as
G. morbillorum
were related to
A. adiacens
. In biochemical tests,
A. elegans
and the seven isolates related to it possessed arginine dihydrolase (ADH) activity but the other
Abiotrophia
species did not. As a result,
A. elegans
strains comprised 8% of the 91 NVS isolates. Our findings suggest that
A. elegans
,
A. adiacens
, and
A. defectiva
exist in the human mouth in proportions of about 1:11:1 and that
A. elegans
can be genetically distinguished from the other two
Abiotrophia
species by PCR-RFLP analysis of 16S rRNA gene sequences and can be biochemically distinguished by ADH activity.
Serological Determination of Hepatitis C Virus Subtypes 1a, 1b, 2a, 2b, 3a, and 4a by a Recombinant Immunoblot Assay
Authors: Schröter Matthias , Feucht Heinz-Hubert , Schäfer Peter , Zöllner Bernhard , Laufs Rainer
Published: December 2019

Serological determination of hepatitis C virus (HCV) subtypes has been hampered by the lack of suitable assays. Therefore, a recombinant immunoblot assay has been established for serological differentiation of HCV subtypes 1a, 1b, 2a, 2b, 3a, and 4a. It consists of recombinant HCV proteins from the NS-4 region propagated in
Escherichia coli
. To confirm the serotyping assay results, the results were compared with those obtained by nucleotide sequencing of the NS-5 region. Sera from 157 patients with chronic HCV infection were examined by this assay, and specific antibodies could be detected in 86% (
n
= 135) of them. The HCV genotype was determined correctly in all but one sample, and the subtypes determined by the serotyping assay corresponded to the HCV subtypes detected by nucleotide sequencing for 95% (
n
= 128) of the samples. These data indicate that HCV subtypes can be distinguished serologically. The assay that is described provides an easier means of identification of infection with different HCV subtypes for wider clinical and epidemiological applications.
Biochemical Identification of
Citrobacter
Species Defined by DNA Hybridization and Description of
Citrobacter gillenii
sp. nov. (Formerly
Citrobacter
Genomospecies 10) and
Citrobacter murliniae
sp. nov. (Formerly
Citrobacter
Genomospecies 11)
Authors: Brenner Don J. , O’Hara Caroline M. , Grimont Patrick A. D. , Janda J. Michael , Falsen Enevold , Aldova Eva , Ageron Elisabeth , Schindler Jiri , Abbott Sharon L. , Steigerwalt Arnold G.
Published: December 2019

Recent work describing six named species and two unnamed genomospecies within
Citrobacter
has enlarged the genus to 11 species. DNA relatedness and phenotypic tests were used to determine how well these species can be identified. One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion. By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level. Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other
Citrobacter
species. Carbon source utilization tests were used to identify 86 of the strains. Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other
Citrobacter
species. Additional strains of
Citrobacter
genomospecies 10 and
Citrobacter
genomospecies 11 were identified, allowing these species to be formally named as
Citrobacter gillenii
sp. nov. and
Citrobacter murliniae
sp. nov., respectively.
Diagnostic Value of Anti-Hepatitis C Virus (HCV) Core Immunoglobulin M in Recurrence of HCV Infection after Orthotopic Liver Transplantation
Authors: Casino Carmela , Lilli Daniela , Rivanera Daniela , Comanducci Antonella , Rossi Massimo , Casciaro Giovanni , Pecorella Irene , Alfani Dario , Mancini Carlo
Published: December 2019

The significance of anti-hepatitis C virus (HCV) core immunoglobulin M (IgM) and its relationship with genotypes, alanine aminotransferase abnormality, and histological data were studied for 18 patients who had undergone orthotopic liver transplantation due to HCV-related end-stage disease. During follow-up, IgM response seemed to be associated with the recurrence of HCV infection but did not correlate with abnormal alanine aminotransferase levels and histological data. In addition, the results of this study indicated that the detection of HCV RNA is critical for diagnosis of reinfection in liver transplantation.
Phylogenetic Analysis of
Rhinosporidium seeberi
’s 18S Small-Subunit Ribosomal DNA Groups This Pathogen among Members of the Protoctistan Mesomycetozoa Clade
Authors: Herr Roger A. , Ajello Libero , Taylor John W. , Arseculeratne Sarath N. , Mendoza Leonel
Published: December 2019

For the past 100 years the phylogenetic affinities of
Rhinosporidium seeberi
have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from
R. seeberi
. Using phylogenetic analysis, by parsimony and distance methods, of
R. seeberi
’s 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (
Dermocystidium
, rossete agent,
Ichthyophonus
, and
Psorospermium
), near the animal-fungal divergence. Our phylogenetic analyses also indicate that
R. seeberi
is the sister taxon of the two
Dermocystidium
species used in this study. This molecular affinity is remarkable since members of the genus
Dermocystidium
form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of
R. seeberi
to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group’s phylogenetic association within the
Eucarya
.
Infection with Agents of Human Granulocytic Ehrlichiosis, Lyme Disease, and Babesiosis in Wild White-Footed Mice (
Peromyscus leucopus
) in Connecticut
Authors: Stafford Kirby C. , Massung Robert F. , Magnarelli Louis A. , Ijdo Jacob W. , Anderson John F.
Published: December 2019

White-footed mice,
Peromyscus leucopus
, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic
Ehrlichia
sp.,
Borrelia burgdorferi
, and
Babesia microti
. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1
Ehrlichia
strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to
B. burgdorferi
, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to
B. microti
by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic
Ehrlichia
sp. organisms were detected in 17 of 23 (73.9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent.
Ehrlichia
spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval
Ixodes scapularis
pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic
Ehrlichia
is transient in most wild
P. leucopus.
Presence of α and
a
Mating Types in Environmental and Clinical Collections of
Cryptococcus neoformans
var.
gattii
Strains from Australia
Authors: Halliday C. L. , Bui T. , Krockenberger M. , Malik R. , Ellis D. H. , Carter D. A.
Published: December 2019

Cryptococcus neoformans
var.
gattii
lives in association with certain species of eucalyptus trees and is a causative agent of cryptococcosis. It exists as two mating types, MATα and MATa, which is determined by a single-locus, two-allele system. In the closely related
C. neoformans
var.
neoformans
, the α mating type has been found to outnumber its a counterpart by at least 30:1, but there have been very limited data on the proportions of each mating type in
C. neoformans
var.
gattii
. In the present study, specific PCR primers were designed to amplify two separate α-mating-type genes from
C. neoformans
var.
gattii
strains. These were used to survey for the presence of the two mating types in clinical and environmental collections of
C. neoformans
var.
gattii
strains from Australia. Sixty-eight of 69 clinical isolates produced both α mating type-specific bands and were assumed to be of the α mating type. The majority of environmental isolates were also of the α mating type, but the a mating type was located in two separate areas. In one area, the a mating type outnumbered the α mating type by 27:2, but in the second area, the ratio of the two mating types was close to the 50:50 ratio expected for sexual recombination.
Onychomycosis Caused by
Blastoschizomyces capitatus
Authors: D’Antonio Domenico , Romano Ferdinando , Iacone Antonio , Violante Beatrice , Fazii Paolo , Pontieri Eugenio , Staniscia Tommaso , Caracciolo Claudia , Bianchini Stefano , Sferra Roberta , Vetuschi Antonella , Gaudio Eugenio , Carruba Giuseppe
Published: December 2019

Blastoschizomyces capitatus
was cultured from the nail of a healthy patient with onychomycosis. The identity of the isolate was initially established by standard methods and ultrastructural analysis and was verified by molecular probing. Strains ATCC 200929, ATCC 62963, and ATCC 62964 served as reference strains for these analyses. To our knowledge, this is the first case of nail infection secondary to paronychia caused by this organism reported in the English literature.
Skin Carriage of Acinetobacters in Hong Kong
Authors: Chu Y. W. , Leung C. M. , Houang E. T. S. , Ng K. C. , Leung C. B. , Leung H. Y. , Cheng A. F. B.
Published: December 2019

We studied the carriage of
Acinetobacter
spp. at five superficial sites in 79 patients from two hospitals, in 133 healthy controls from the community (medical students and new nurses), and in 198 student nurses in different classes. A total of 431 isolates from 364 positive sites of 201 subjects and 124 blood culture isolates (1997 to 1998) were genospeciated by amplified ribosomal DNA restriction analysis. Genospecies 3 was the most common species. The carriage rate of student nurses (42 of 131) was significantly lower than that of new nurses from the community (25 of 38) (chi-square test,
P
= 0.0004; odds ratio [OR], 4.08; 95% confidence limits, 1.78 to 9.41) but not significantly different (
P
= 0.1) from that of patients in the same hospital (20 of 42). Genospecies from blood cultures and subjects (acute patients and student nurses) from Prince of Wales Hospital were similar to one another but different from subjects from the community or from another hospital (chi-square test,
P
< 0.0001). Half of the subjects who were positive at at least two sites had different genospecies. Of the 28 sites examined, 68% showed strain variation among isolates of the same genospecies by random amplified polymorphic DNA analysis. Half of the 106 subjects who had samples taken again within 6 weeks or 6 months later were positive only once. In the 17 subjects who were positive on at least two occasions, each occasion yielded different genospecies in 13 subjects. Our results indicate that skin carriage in the majority of healthy subjects is characterized by low density, variation in genospecies and strains, short-term duration, and the typicality of a given locality.
Inter- and Intralaboratory Comparison of
Ehrlichia equi
and Human Granulocytic Ehrlichiosis (HGE) Agent Strains for Serodiagnosis of HGE by the Immunofluorescent-Antibody Test
Authors: Walls Jennifer J. , Aguero-Rosenfeld Maria , Bakken Johan S. , Goodman Jesse L. , Hossain Deloar , Johnson Russell C. , Dumler J. Stephen
Published: December 2019

Human granulocytic ehrlichiosis (HGE) is usually diagnosed by immunofluorescent antibody (IFA) serology with
Ehrlichia equi
-infected neutrophils or HGE agent-infected cultured HL60 cells. The HGE agent and
E. equi
are antigenically diverse, and interpretation of serologic results is also often variable. Thus, we investigated the sensitivity and specificity of various HGE agent and
E. equi
antigens used for IFA diagnosis by three different laboratories. Serum samples from 28 patients with well-characterized HGE and 9 patients with suspected HGE who were investigated by PCR, blood smear examinations, and serology were used, along with 9 serum samples from patients with other rickettsial and ehrlichial infections. Each serum sample was tested with up to 10 different antigen preparations. Overall, qualitative IFA results agreed in 70% of the samples. Titers among antigens were similar (
r
= 0.89 to 0.96), but titers of individual samples varied by fourfold or more in 5 of 81 (6%) of the serum samples. Sensitivity ranged from 100% to 82%, and specificity varied from 100% to 67%, but these differences were not significant, even among those tested in the same laboratory or between two different laboratories. Antibodies were detected in 14 to 44% of acute-phase sera from confirmed HGE patients. Most false-positive reactions resulted with
Ehrlichia chaffeensis
; when these sera were excluded, the specificity of most antigens was 91 to 100%. These data indicate that IFA results often agree and that IFA is useful for diagnosis of HGE in convalescence. However, without further standardization, variability among serologic tests using
E. equi
and HGE agent isolates for diagnosis of HGE will occasionally provide discrepant results and confound diagnosis.
Cocirculation of Two Distinct Lineages of Equine Influenza Virus Subtype H3N8
Authors: Oxburgh L. , Klingeborn B.
Published: December 2019

Direct amplification and sequencing of the hemagglutinin (HA) genes of equine influenza virus subtype H3N8 was undertaken in order to characterize strains of this virus circulating in Sweden. The majority of viruses from outbreaks during 1997 analyzed belonged to the American lineage of H3 equine influenza, and one strain was shown to belong to the European lineage. Furthermore, it was shown that recent American-lineage strains are mutated at amino acid position 190 of the HA during serial passage in embryonated hens’ eggs. Host cell adaptation of these viruses thus takes place at antigenic region B of the HA.
Comparison of Commercial Amies Transport Systems with In-House Amies Medium for Recovery of
Neisseria gonorrhoeae
Authors: Thompson Donna S. , French Susanne A.
Published: December 2019

Microbiologists are still encumbered by the variable performance of Amies charcoal transport medium in recovery of
Neisseria gonorrhoeae
. The objective of this study was to evaluate and select a good quality commercial system to replace our in-house preparation. We adsorbed 0.1 ml of a suspension from 30 gonococcal isolates onto each swab type and replaced the swab into the transport medium. We plated the swabs to New York City medium at 0, 24, 48, 72, and 96 h. We compared the survival of each isolate in the commercial Amies transport systems with that in our in-house Amies transport medium. The best recovery was observed with Copan transport systems. Some systems are inadequate and unacceptable for culture of gonococci.
Isolation of
Providencia heimbachae
from Human Feces
Authors: Mohr O’Hara Caroline , Steigerwalt Arnie G. , Green Donna , McDowell Michael , Hill Bertha C. , Brenner Don J. , Miller J. Michael
Published: December 2019

Providencia heimbachae
was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of
P. heimbachae
from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of
P. heimbachae.
Identification of and Hydrogen Peroxide Production by Fecal and Vaginal Lactobacilli Isolated from Japanese Women and Newborn Infants
Authors: Song Yu-Li , Kato Naoki , Matsumiya Yoshiko , Liu Cheng-Xu , Kato Haru , Watanabe Kunitomo
Published: December 2019

We identified
Lactobacillus
isolates from Japanese women and newborn infants by a DNA-DNA hybridization method. The predominating lactobacilli were
Lactobacillus crispatus
and
Lactobacillus gasseri
in the women’s vaginas and the newborns’ intestines and
L. gasseri
and
Lactobacillus fermentum
in the women’s intestines. All
L. crispatus
strains were exclusively strong H
2
O
2
producers.
Third Human Isolate of a
Desulfovibrio
sp. Identical to the Provisionally Named
Desulfovibrio fairfieldensis
Authors: La Scola Bernard , Raoult Didier
Published: December 2019

Desulfovibrio fairfieldensis
was isolated from the urine sample of a patient with a urinary tract infection and meningoencephalitis. It was identified by 16S rRNA gene amplification and sequencing.
Diarrheagenic
Escherichia coli
and Acute and Persistent Diarrhea in Returned Travelers
Authors: Schultsz C. , van den Ende J. , Cobelens F. , Vervoort T. , van Gompel A. , Wetsteyn J. C. F. M. , Dankert J.
Published: December 2019

To determine the role of diarrheagenic
Escherichia coli
in acute and persistent diarrhea in returned travelers, a case control study was performed. Enterotoxigenic
E. coli
(ETEC) was detected in stool samples from 18 (10.7%) of 169 patients and 4 (3.7%) of 108 controls. Enteroaggregative
E. coli
(EAggEC) was detected in 16 (9.5%) patients and 7 (6.5%) controls. Diffuse adherent
E. coli
strains were commonly present in both patients (13%) and controls (13.9).
Campylobacter
and
Shigella
species were the other bacterial enteropathogens most commonly isolated (10% of patients, 2% of controls). Multivariate analysis showed that the presence of ETEC was associated with acute diarrhea (odds ratio [OR], 6.7; 95% confidence interval [CI], 1.5 to 29.1;
P
= 0.005), but not with persistent diarrhea (OR, 1.6; 95% CI, 0.4 to 7.4). EAggEC was significantly more often present in patients with acute diarrhea than in controls (
P
= 0.009), but no significant association remained after multivariate analysis. ETEC and EAggEC are frequently detected in returned travelers with diarrhea. The presence of ETEC strains is associated with acute but not with persistent diarrhea.
Brachyspira aalborgi
Infection Diagnosed by Culture and 16S Ribosomal DNA Sequencing Using Human Colonic Biopsy Specimens
Authors: Kraaz Wolfgang , Pettersson Bertil , Thunberg Ulf , Engstrand Lars , Fellström Claes
Published: December 2019

In this study we report on the isolation and characterization of the intestinal spirochete
Brachyspira aalborgi
using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 μg of spectinomycin/ml and 5 μg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of
B. aalborgi
. A similarity of 99.7% in the nucleotide sequence was found between W1 and 513A
T
, based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513A
T
in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate
B. aalborgi
.
Detection of Trichomonosis in Vaginal and Urine Specimens from Women by Culture and PCR
Authors: Lawing Lisa F. , Hedges Spencer R. , Schwebke Jane R.
Published: December 2019

Vaginal trichomonosis is a highly prevalent infection which has been associated with human immunodeficiency virus acquisition and preterm birth. Culture is the current “gold standard” for diagnosis. As urine-based testing using DNA amplification techniques becomes more widely used for other sexually transmitted diseases (STDs) such as gonorrhea and chlamydia, a similar technique for trichomonosis would be highly desirable. Women attending an STD clinic for a new complaint were screened for
Trichomonas vaginalis
by wet-preparation (wet-prep) microscopy and culture and for the presence of
T. vaginalis
DNA by specific PCR of vaginal and urine specimens. The presence of trichomonosis was defined as the detection of
T. vaginalis
by direct microscopy and/or culture from either vaginal samples or urine. The overall prevalence of trichomonosis in the population was 28% (53 of 190). The sensitivity and specificity of PCR using vaginal samples were 89 and 97%, respectively. Seventy-four percent (38 of 51) of women who had a vaginal wet prep or vaginal culture positive for trichomonads had microscopic and/or culture evidence of the organisms in the urine. Two women were positive for trichomonads by wet prep or culture only in the urine. The sensitivity and specificity of PCR using urine specimens were 64 and 100%, respectively. These results indicate that the exclusive use of urine-based detection of
T. vaginalis
is not appropriate in women. PCR-based detection of
T. vaginalis
using vaginal specimens may provide an alternative to culture.
Evaluation of Amplified Fragment Length Polymorphism Analysis for Inter- and Intraspecific Differentiation of
Mycobacterium bovis
,
M. tuberculosis
, and
M. ulcerans
Authors: Huys G. , Rigouts L. , Chemlal K. , Portaels F. , Swings J.
Published: December 2019

The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrimination of
Mycobacterium bovis
(17 strains),
M. tuberculosis
(15 strains), and
M. ulcerans
(12 strains) at the inter- and intraspecific level. The AFLP technique is a whole-genome coverage genotypic fingerprinting method based on the selective PCR amplification of modified restriction fragments obtained through a double enzymatic digest and subsequent ligation of double-stranded restriction site-specific adapter oligonucleotides. Selective amplification of
Apa
I/
Taq
I templates with primer combination A02-T02 (both having an additional C at their 3′ end) generated autoradiographic AFLP fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing clear separation of
M. ulcerans
(cluster I) from the
M. tuberculosis
complex members
M. bovis
and
M. tuberculosis
(cluster II). Calculation of similarities using the band-based Dice correlation coefficient instead of the Pearson product-moment correlation coefficient revealed a further subgrouping in cluster II. The two resulting subclusters corresponded with the phenotypic identity of
M. bovis
and
M. tuberculosis
, respectively, and could also be visually identified by two AFLP marker bands. Because of the relatively low degree of genotypic variation among the AFLP band patterns of the latter two taxa, no correlation could be found with previously reported molecular typing data or with geographical origin. The use of primer combination A02-T01 (the latter having an A as selective base) did not increase the resolving power within the
M. tuberculosis
complex but resulted in a visual subgrouping of the
M. ulcerans
strains that was not observed with primer combination A02-T02. Based on the presence or absence of a single AFLP marker band, the
M. ulcerans
isolates could be unambiguously classified in two continental types corresponding with the African and Australian origin of the strains, respectively. In conclusion, the radioactive AFLP method proved to be a reproducible and reliable taxonomic tool for the differentiation of the three mycobacterial species under study and also demonstrated its potential use for typing of
M. ulcerans
strains when employing multiple primer combinations.
Differentiation between
Candida dubliniensis
and
Candida albicans
by Fatty Acid Methyl Ester Analysis Using Gas-Liquid Chromatography
Authors: Peltroche-Llacsahuanga Heidrun , Schmidt Silke , Seibold Michael , Lütticken Rudolf , Haase Gerhard
Published: December 2019

Candida dubliniensis
is often found in mixed culture with
C. albicans
, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of
C. dubliniensis
is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (
n
= 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (
C. albicans
,
n
= 32;
C. dubliniensis
,
n
= 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for
C. albicans
and
C. dubliniensis
ranged from 0.11 to 0.96 and 0.53 to 0.93 (for all but one), respectively. Only three isolates of
C. albicans
(9.4%) were misidentified as
C. dubliniensis
, whereas all isolates of
C. dubliniensis
were correctly identified. Resulting differentiation accuracy was 90.6% for
C. albicans
and 100% for
C. dubliniensis
. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.
Two Enzyme Immunoassays and PCR for Detection of
Helicobacter pylori
in Stool Specimens from Pediatric Patients before and after Eradication Therapy
Authors: Makristathis Athanasios , Barousch Wolfgang , Pasching Eva , Binder Christa , Kuderna Christa , Apfalter Petra , Rotter Manfred L. , Hirschl Alexander M.
Published: December 2019

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect
Helicobacter pylori
antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine
H. pylori
-infected children with dyspepsia received eradication therapy. Successful treatment was determined by a negative [
13
C]urea breath test 4 and 12 weeks after discontinuation of therapy. Fecal specimens were collected prior to eradication therapy as well as 4 weeks after the end of treatment. Successfully treated children delivered stool samples at 6, 8, and 12 weeks posttreatment also. Specimens were examined by seminested PCR and Premier Platinum HpSA and were reexamined by both EIAs as soon as FemtoLab H. pylori was available. In the first test series, the overall sensitivities of PCR and Premier Platinum HpSA were 93.0 and 91.1%, respectively. With specimens collected at 4 weeks after treatment, the respective specificities were 68.8 and 79.3%. After longer follow-up periods, however, they gradually increased to 100 and 96.9%, respectively. In the new test series, Premier Platinum HpSA delivered a considerably lower number of false-positive results (4 versus 18), indicating intertest variations. The overall test sensitivity was 94.6%, and the overall specificity was 97.5%. FemtoLab H. pylori showed an excellent performance with an overall sensitivity and specificity of 98.2 and 98.1%, respectively. Thus, in contrast to PCR, both EIAs were shown to be suitable for early posttreatment control.
Utilization of Exocellular Mannan from
Rhodotorula glutinis
as an Immunoreactive Antigen in Diagnosis of Leptospirosis
Authors: Matsuo Kouki , Isogai Emiko , Araki Yoshio
Published: December 2019

Previously,
Rhodotorula glutinis
was reported to produce a large amount of exocellular mannan, having a repeating unit of →3)-
d
-Man
p
-(1→4)-
d
-Man
p
-(1→. Recently, we found that antigenic polysaccharides of
Leptospira biflexa
serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of
Rhodotorula
mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from
R. glutinis
AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and
1
H- and
13
C-labeled nuclear magnetic resonance spectrometry, the
Rhodotorula
mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the
Rhodotorula
mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis.
Evaluation of a PCR Primer Based on the Isocitrate Dehydrogenase Gene for Detection of
Helicobacter pylori
in Feces
Authors: Argyros Frauke C. , Ghosh Mayurika , Huang Lili , Masubuchi Naoko , Cave David R. , Grübel Peter
Published: December 2019

In order to improve detection and identification of
Helicobacter pylori
in highly contaminated samples, we evaluated new specific primers based on the DNA base sequence within the isocitrate dehydrogenase (
icd
) gene to amplify a 1,200-bp DNA segment. The specificity of the
icd
primer was tested against DNA derived from various bacteria, including 7
Helicobacter
species and a panel of 1 gram-variable, 2 gram-positive, and 16 gram-negative bacteria, as well as DNA from houseflies and feces from
H. pylori
-negative patients. The primers permitted the detection of all clinical
H. pylori
isolates tested, but no reactions were observed with negative controls. Several procedures for DNA extraction from feces were evaluated using PCR with
icd
primers. The lower limits of detection of
H. pylori
DNA from two different sources containing the same number of
H. pylori
organisms, a pure culture and feces spiked with
H. pylori
, were established for each extraction method tested. The results were 8.0 × 10
3
CFU/ml for cultures of pure
H. pylori
, and 8.0 × 10
6
CFU/ml for
H. pylori
from feces, using the phenol-chloroform method; 8.0 × 10
2
and 7.0 × 10
3
CFU/ml, respectively, for a glass matrix and chaotropic solution protocol; 8.0 × 10
2
and 7.0 × 10
3
CFU/ml, respectively, for the QIAamp tissue kit; and 5.0 × 10
2
and 5.0 × 10
3
CFU/ml, respectively, for the XTRAX DNA extraction kit. We conclude that the use of the
icd
gene as a primer for PCR represents a specific and sensitive assay for detection of
H. pylori
in highly contaminated samples.
Bacillus anthracis
Diversity in Kruger National Park
Authors: Smith K. L. , DeVos V. , Bryden H. , Price L. B. , Hugh-Jones M. E. , Keim P.
Published: December 2019

The Kruger National Park (KNP), South Africa, has a recorded history of periodic anthrax epidemics causing widespread disease among wild animals.
Bacillus anthracis
is the causative agent of anthrax, a disease primarily affecting ungulate herbivores. Worldwide there is little diversity among
B. anthracis
isolates, but examination of variable-number tandem repeat (VNTR) loci has identified six major clones, with the most dissimilar types split into the A and B branches. Both the A and B types are found in southern Africa, giving this region the greatest genetic diversity of
B. anthracis
worldwide. Consequently, southern Africa has been hypothesized to be the geographic origin of
B. anthracis
. In this study, we identify the genotypic types of 98 KNP
B. anthracis
isolates using multiple-locus VNTR analysis. Two major types are evident, the A branch and the B branch. The spatial and temporal distribution of the different genotypes indicates that anthrax epidemic foci are independent, though correlated through environmental cues. Kruger B isolates were found on significantly higher-calcium and higher-pH soils than were Kruger type A. This relationship between genotype and soil chemistry may be due to adaptive differences among divergent anthrax strains. While this association may be simply fortuitous, adaptation of A types to diverse environmental conditions is consistent with their greater geographic dispersal and genetic dissimilarity.
Comparison between Plasma and Whole Blood Specimens for Detection of
Aspergillus
DNA by PCR
Authors: Loeffler Juergen , Hebart Holger , Brauchle Ulrike , Schumacher Ulrike , Einsele Hermann
Published: December 2019

Ninety-six plasma and whole blood specimens from nine selected patients were analyzed for the presence of
Aspergillus
DNA. Nineteen specimens from three patients with proven aspergillosis were PCR positive in both materials, whereas an additional 22 were PCR positive in whole blood only. All 36 samples from six patients without signs of aspergillosis were negative in both assays. We conclude that although plasma and whole blood spiked with
Aspergillus
conidia showed an identical lower detection limit (10 CFU), the sensitivity of plasma PCR was lower than that of PCR performed on whole blood samples.
Isolation of
Moraxella canis
from an Ulcerated Metastatic Lymph Node
Authors: Vaneechoutte Mario , Claeys Geert , Steyaert Sophia , De Baere Thierry , Peleman Renaat , Verschraegen Gerda
Published: December 2019

Moraxella canis
was isolated in large numbers from an ulcerated supraclavicular lymph node of a terminal patient, who died a few days later. Although the patient presented with septic symptoms and with a heavy growth of gram-negative diplococci in the lymph node, blood cultures remained negative.
M. canis
is an upper-airway commensal from dogs and cats and is considered nonpathogenic for humans, although this is the third reported human isolate of this species.
Mycobacterium branderi
from Both a Hand Infection and a Case of Pulmonary Disease
Authors: Wolfe Joyce , Turenne Christine , Alfa Michelle , Harding Godfrey , Thibert Louise , Kabani Amin
Published: December 2019

Mycobacterium branderi
, a potential human pathogen first characterized in 1995, has been isolated from respiratory tract specimens. We report here a case in which
M. branderi
was the only organism isolated upon culture from a hand infection. This isolate, along with a second isolate from a bronchial specimen, was subjected to conventional identification tests for mycobacterial species. Further analysis by high-performance liquid chromatography (HPLC) of mycolic acids and 16S rRNA gene sequencing was performed, and the antibiotic susceptibility profile was determined for both strains. Biochemical tests and the HPLC pattern were consistent with that of
M. branderi
and
M. celatum
, which are very similar. The 16S rRNA gene sequence of both strains corresponded to that of
M. branderi
and enabled us to confidently differentiate this organism from other closely related species such as
M. celatum
. This contributes to a further understanding of the status of this species as a potential human pathogen as well as illustrating the need for molecular diagnostics as a complementary method for the identification of rare mycobacterial species.
Improved Immunodiagnosis of Cystic Hydatid Disease by Using a Synthetic Peptide with Higher Diagnostic Value Than That of Its Parent Protein,
Echinococcus granulosus
Antigen B
Authors: González-Sapienza Gualberto , Lorenzo Carmen , Nieto Alberto
Published: December 2019

The assays are used for the diagnosis of hydatid disease are still imperfect. The reported diagnostic sensitivity and specificity vary greatly depending on the panel of sera used, the laboratory conducting the assay, and, more critically, the antigen used. To contribute to its standardization, we have recently ranked the diagnostic performances of the major parasite antigens and the available synthetic peptides using a large collection of serum samples. That work showed that antigen B (AgB) possesses the highest diagnostic value among these antigens. In the present work we further dissected its antigenicity by analyzing the reactivity of the same panel of sera against a set of synthetic peptides spanning the sequence of both AgB subunits. The N-terminal extension of these subunits appeared to be immunodominant in human infections. A 38-mer peptide (p176) delineated from the N-terminal extension of the AgB/1 subunit performed in an enzyme-linked immunosorbent assay with a higher diagnostic sensitivity (80%) and specificity (94%) than native AgB, Ag5, or any other peptide antigen tested against this collection of serum samples. In view of its high diagnostic value and its nature as a well-defined reproducible antigen, p176 could conveniently be used as a reference standard antigen in the diagnosis of hydatid disease.
Evaluation of Fluorescence-Based Amplified Fragment Length Polymorphism Analysis for Molecular Typing in Hospital Epidemiology: Comparison with Pulsed-Field Gel Electrophoresis for Typing Strains of Vancomycin-Resistant
Enterococcus faecium
Authors: Antonishyn Nick A. , McDonald Ryan R. , Chan Edward L. , Horsman Greg , Woodmansee Carla E. , Falk Pamela S. , Mayhall C. Glen
Published: December 2019

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the “gold standard” for molecular typing in epidemiology.
Detection and Identification of Mycobacteria by Amplification of the Internal Transcribed Spacer Regions with Genus- and Species-Specific PCR Primers
Authors: Park Heekyung , Jang Hyunjung , Kim Cheolmin , Chung Byungseon , Chang Chulhun L. , Park Soon Kew , Song Sundae
Published: December 2019

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118
Mycobacterium tuberculosis
isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the
M. tuberculosis
complex-specific primers amplified an approximately 120-bp fragment only for the
M. tuberculosis
complex. Multiplex PCR with the panmycobacterial primers and the
M. tuberculosis
complex-specific primers amplified two fragments that were specific for all mycobacteria and the
M. tuberculosis
complex, respectively. PCR with
M. avium
complex-,
M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-
, and
M. szulgai
-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.
Mycobacterium heckeshornense
sp. nov., a New Pathogenic Slowly Growing
Mycobacterium
sp. Causing Cavitary Lung Disease in an Immunocompetent Patient
Authors: Roth Andreas , Reischl Udo , Schönfeld Nicolas , Naumann Ludmila , Emler Stefan , Fischer Marga , Mauch Harald , Loddenkemper Robert , Kroppenstedt Reiner M.
Published: December 2019

A pathogenic scotochromogenic
Mycobacterium xenopi
-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making two surgical interventions necessary after years of chronic disease. This case prompted us to characterize this mycobacterium by a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers which were typical for the genus
Mycobacterium
, i.e., the
meso
isomer of 2,6-diaminopimelic acid, arabinose, and galactose as diagnostic whole-cell sugars, MK-9(H
2
) as the principal isoprenoid quinone, a mycolic acid pattern of α-mycolates, ketomycolates, and wax ester mycolates, unbranched saturated and unsaturated fatty acids plus a significant amount of tuberculostearic acid, and small amounts of a C
20:0
secondary alcohol. On the basis of its unique 16S rRNA and 16S-23S spacer gene sequences, we propose that the isolate should be assigned to a new species,
Mycobacterium heckeshornense
. This novel species is phylogenetically closely related to
M. xenopi
. The type strain of
M. heckeshornense
is strain S369 (DSM 44428
T
). The GenBank accession number of the 16S rRNA gene of
M. heckeshornense
is
AF174290
.
PCR Detection of
Escherichia coli
O157:H7 Directly from Stools: Evaluation of Commercial Extraction Methods for Purifying Fecal DNA
Authors: Holland J. L. , Louie L. , Simor A. E. , Louie M.
Published: December 2019

Rapid identification of
Escherichia coli
O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract
E. coli
O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the
E. coli
O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.