bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of a Nonradiometric Liquid-Medium Method (MB REDOX) with the BACTEC System for Growth and Identification of Mycobacteria in Clinical Specimens
Authors: Liu Yung-Ching , Huang Tsi-Shu , Huang Wen-Kuei
Published: December 2019

Early identification of tuberculosis in the clinical setting is of great importance in order for specific therapy to be swiftly initiated. MB REDOX (Heipha Diagnostika), a growth-based medium without radioactive materials, was evaluated and was compared to the BACTEC system for detection of mycobacteria, including the
Mycobacterium tuberculosis
complex and atypical mycobacteria. MB REDOX consists of a Kirchner medium enriched with growth-promoting additives, antibiotic compounds, and a redox indicator which can be monitored to detect growth of mycobacteria with the naked eye. MB REDOX only detects growth and cannot differentiate the
M. tuberculosis
complex (
M. tuberculosis
,
M. bovis
, and
M. africanum
) from other species of
Mycobacterium
. Therefore, PCR-restriction fragment length polymorphism analysis (PRA) was used in this investigation to identify to the species level organisms showing positive growth with MB REDOX. Our data demonstrate the usefulness of MB REDOX for the detection of mycobacteria in clinical specimens. The rate of detection of
M. tuberculosis
complex with MB REDOX (84.3%) was higher than that with the BACTEC system (68.6%). When combined with PRA for species identification, MB REDOX is easy to perform and is suited to most clinical laboratory settings for the detection and identification of mycobacteria.
Prevalence of the Amylase-Binding Protein A Gene (
abpA
) in Oral Streptococci
Authors: Brown Alan E. , Rogers Jeffrey D. , Haase Elaine M. , Zelasko Peter M. , Scannapieco Frank A.
Published: December 2019

Salivary amylase binds specifically to a number of oral streptococcal species. This interaction may play an important role in dental plaque formation. Recently, a 585-bp gene was cloned and sequenced from
Streptococcus gordonii
Challis encoding a 20.5-kDa amylase-binding protein (AbpA). The goal of this study was to determine if related genes are present in other species of oral streptococci. Biotinylated
abpA
was used in Southern blot analysis to screen genomic DNA from several strains representing eight species of oral streptococci. This probe hybridized with a 4.0-kb
Hin
dIII restriction fragment from all 13 strains of
S. gordonii
tested. The probe did not appear to bind to any restriction fragments from other species of amylase-binding oral streptococci including
Streptococcus mitis
(with the exception of 1 of 14 strains),
Streptococcus crista
(3 strains),
Streptococcus anginosus
(1 strain), and
Streptococcus parasanguinis
(1 strain), or to non-amylase-binding oral streptococci including
Streptococcus sanguinis
(3 strains),
Streptococcus oralis
(4 strains), and
Streptococcus mutans
(1 strain). Primers homologous to sequences within the 3′ and 5′ ends of
abpA
yielded products of 400 bp following PCR of genomic DNA from the Southern blot-positive strains. Several of these PCR products were cloned and sequenced. The levels of similarity of these cloned products to the
abpA
of
S. gordonii
Challis ranged from 91 to 96%. These studies reveal that the
abpA
gene appears to be specific to
S. gordonii
and differs from genes encoding amylase-binding proteins from other species of amylase-binding streptococci.
Evaluation of Three Commercial Serological Tests with Different Methodologies To Assess
Helicobacter pylori
Infection
Authors: van der Ende A. , van der Hulst R. W. M. , Roorda P. , Tytgat G. N. J. , Dankert J.
Published: December 2019

The sera of 142
Helicobacter pylori
-positive and 32
H. pylori
-negative patients were assessed by a desktop test (QuickVue), an enzyme-linked immunosorbent assay (ELISA) (HM-CAP), and a solid-phase, two-step chemiluminescent enzyme immunoassay (Immulite). These tests yielded sensitivities of 97, 97, and 91% and specificities of 97, 94, and 100%, respectively. In conclusion, the desktop test and the ELISA are more sensitive than the chemiluminescent enzyme immunoassay (
P
< 0.05). The chemiluminescent enzyme immunoassay has the advantage that it is fully automated.
Evaluation of a Rapid Assay for Detection of
Chlamydia trachomatis
Infections in Outpatient Clinics in South Kalimantan, Indonesia
Authors: Widjaja Susana , Cohen Surekha , Brady William E. , O'reilly Kevin , Susanto  , Wibowo Ajar , Cahyono  , Graham Robert R. , Porter Kevin R.
Published: December 2019

A multicenter cross-sectional survey was conducted comparing a commercially available chlamydial optical immunoassay (OIA) to the chlamydial ligase chain reaction (LCR). Endocervical samples from 415 outpatients visiting clinics from three hospitals in South Kalimantan, Indonesia, were evaluated. Relative to the LCR, the overall sensitivity and specificity of the OIA were 31.6 and 98.9%, respectively. The sensitivity of the OIA varied among the three hospital laboratories, ranging from 20 to 50%. The OIA performance was slightly lower on samples from patients attending dermatovenereology clinics than on samples from nondermatovenereology clinic patients. The results indicate that the OIA did not perform well compared to LCR.
Identification of
Hafnia alvei
with the MicroScan WalkAway System
Authors: Rodríguez L. A. , Vivas J. , Gallardo C. S. , Acosta F. , Barbeyto L. , Real F.
Published: December 2019

Hafnia alvei
is a gram-negative facultatively anaerobic bacillus that belongs to the family
Enterobacteriaceae
. This organism is a causative agent of intestinal disorders and is found in different environments.
H. alvei
has received increased clinical attention as a cause of different infections in humans. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identification of 21
H. alvei
strains. The MicroScan WalkAway system was found capable of correctly identifying 20 of the 21 strains tested.
Quantitative Detection of Hepatitis B Virus by Transcription-Mediated Amplification and Hybridization Protection Assay
Authors: Kamisango Keiichi , Kamogawa Chieko , Sumi Mayumi , Goto Susumu , Hirao Akihide , Gonzales Frank , Yasuda Kiyomi , Iino Shiro
Published: December 2019

We have developed a sensitive and quantitative assay using transcription-mediated amplification and hybridization protection assay for the detection of hepatitis B virus (HBV) DNA in serum. The transcription-mediated amplification was carried out in a single tube. The hybridization protection assay was carried out in a microtiter plate with two probes with different specific activities to obtain a broad detection range. As a result, the assay had a detection range of 5 × 10
3
to 5 × 10
8
genome equivalents (GE)/ml and good quantitative accuracy on a logarithmic scale. A moderately sized manual assay run can be completed within 5 h. Measurements of the amounts of HBV DNA in clinical samples by the assay showed the amounts under various disease conditions to be widely distributed (more than 5 logs, from approximately 5 × 10
3
to 5 × 10
8
GE/ml). It was also shown that the amount of HBV DNA in one chronic hepatitis patient varied widely, with a range of more than 5 logs during long-term monitoring. Our assay has the potential to be used to monitor and determine the prognosis of HBV patients and carriers, especially during interferon treatment.
Molecular Genotyping of
Staphylococcus aureus
Strains: Comparison of Repetitive Element Sequence-Based PCR with Various Typing Methods and Isolation of a Novel Epidemicity Marker
Authors: van der Zee Anneke , Verbakel Harold , van Zon Johan-Carlo , Frenay Ine , van Belkum Alex , Peeters Marcel , Buiting Anton , Bergmans Anneke
Published: December 2019

Repetitive sequence-based (Rep)-PCR genotyping as described here is based on the presence of homologues of
Mycoplasma pneumoniae
repeat-like elements in
Staphylococcus
. In this study we comparatively evaluated the usefulness of rep-PCR typing with two sets of well-defined collections of
Staphylococcus aureus
strains. Rep-PCR analysis of the first collection of
S. aureus
strains (
n
= 59) and one
Staphylococcus intermedius
strain showed 14 different rep-PCR patterns, with each pattern harboring 6 to 15 DNA fragments. The discriminatory power of rep-PCR typing compared well to those of arbitrarily primed PCR (average of 20 types) and pulsed-field gel electrophoresis (11 types).
S. aureus
strain collection I comprised four outbreak-related groups of isolates. The isolates in only one group were found to have identical rep-PCR profiles. However, in an analysis of isolates from three additional independent local outbreaks (
n
for outbreaks 1 and 2 = 5,
n
for outbreak 3 = 12), identical rep-PCR types were found among strains isolated during each outbreak. Therefore, we conclude that rep-PCR genotyping may be an easy and fast method for monitoring of the epidemiology of nosocomial
Staphylococcus
infections. Rep-PCR analysis of strain collection II, which consisted of epidemic and nonepidemic methicillin-resistant
S. aureus
(MRSA) strains, revealed that a cluster of similar rep-PCR profiles was found among MRSA isolates which were more frequently isolated and which were most often associated with outbreaks.
Rapid Method for Screening Dried Blood Samples on Filter Paper for Human Immunodeficiency Virus Type 1 DNA
Authors: Panteleeff Dana DeVange , John Grace , Nduati Ruth , Mbori-Ngacha Dorothy , Richardson Barbra , Kreiss Joan , Overbaugh Julie
Published: December 2019

PCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1gagDNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes.
Prevalence of β2-ToxigenicClostridium perfringensin Horses with Intestinal Disorders
Authors: Herholz Cornelia , Miserez Raymond , Nicolet Jacques , Frey Joachim , Popoff Michel , Gibert Maryse , Gerber Heinz , Straub Reto
Published: December 2019

The incidence of a new, yet unassigned toxin type ofClostridium perfringenscontaining the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically.C. perfringensisolates were typed for the presence of the α-, β-, β2-, and ɛ-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin genecpb2. β2-ToxigenicC. perfringenswas detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenicC. perfringens. No β2-toxigenicC. perfringenswas found in the samples from the 58 control horses, of which only one fecal sample containedC. perfringenstype A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenicC. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenicC. perfringensand lethal progression of the disease. NoC. perfringensstrains isolated in this study contained genes for the β- and ɛ-toxins and enterotoxin. The high incidence of β2-toxigenicC. perfringensin samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that β2-toxigenicC. perfringensplay an important role in the pathogenesis of typhlocolitis.
Relatedness of
Streptococcus suis
Serotype 2 Isolates from Different Geographic Origins as Evaluated by Molecular Fingerprinting and Phenotyping
Authors: Chatellier Sonia , Gottschalk Marcelo , Higgins Robert , Brousseau Roland , Harel Josee
Published: December 2019

The genetic diversity of 88
Streptococcus suis
serotype 2 isolates which were recovered from various countries was examined by randomly amplified polymorphic DNA (RAPD) analysis with three primers. This bacterial collection included 80 isolates of porcine origin and 8 of human origin. This investigation allowed the identification of 23 RAPD types containing 1 to 30 isolates originating from one to six countries. Common RAPD patterns were found between human and pig isolates. The isolates were also tested for the production of virulent factors such as hemolysin, muramidase-released protein (MRP), and extracellular factor (EF). All isolates exhibiting the virulent phenotype hemolysin
+
MRP
+
EF
+
clearly clustered on the basis of fingerprinting by RAPD analysis. In a similar way, most of isolates with the hemolysin
−
MRP
−
EF
−
phenotype were assigned to one RAPD cluster. Therefore, RAPD clusters are more related to the phenotype defined with hemolysin, MRP, and EF than to the geographic origin of the isolates. These data indicate that RAPD analysis used in conjunction with phenotypic methods provides a reliable method for the assessment of the clonal relationship between
S. suis
isolates responsible for infections in pigs or humans, especially for those exhibiting the classic “virulent” phenotype hemolysin
+
MRP
+
EF
+
.
Performance Characteristics of an Enzyme-Linked Immunosorbent Assay for Determining Salivary Immunoglobulin G Response to
Helicobacter pylori
Authors: De Pascalis R. , Del Pezzo M. , Nardone G. , Budillon G. , Lavitola A.
Published: December 2019

We evaluated the salivary immunoglobulin G (IgG) immune response to
Helicobacter pylori
in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive
H. pylori
culture showed significantly higher titers of antibodies than subjects with no detectable
H. pylori
: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-
H. pylori
IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.
PCR Targeted to the 16S-23S rRNA Gene Intergenic Spacer Region of
Clostridium difficile
and Construction of a Library Consisting of 116 Different PCR Ribotypes
Authors: Stubbs Simon L. J. , Brazier Jon S. , O’Neill Gael L. , Duerden Brian I.
Published: December 2019

A reference library of types of
Clostridium difficile
has been constructed by PCR ribotyping isolates (
n
= 2,030) from environmental (
n
= 89), hospital (
n
= 1,386), community practitioner (
n
= 395), veterinary (
n
= 27), and reference (
n
= 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7.5% of community infections.
Genetic Diversity of
Borrelia burgdorferi
in Lyme Disease Patients as Determined by Culture versus Direct PCR with Clinical Specimens
Authors: Liveris Dionysios , Varde Shobha , Iyer Radha , Koenig Seth , Bittker Susan , Cooper Denise , McKenna Donna , Nowakowski John , Nadelman Robert B. , Wormser Gary P. , Schwartz Ira
Published: December 2019

Two hundred seventeen isolates of
Borrelia burgdorferi
originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of
B. burgdorferi
performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [
P
< 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of
B. burgdorferi
patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.
Nested Duplex PCR To Detect
Bordetella pertussis
and
Bordetella parapertussis
and Its Application in Diagnosis of Pertussis in Nonmetropolitan Southeast Queensland, Australia
Authors: Farrell D. J. , Daggard G. , Mukkur T. K. S.
Published: December 2019

A duplex PCR to detect
Bordetella pertussis
and
Bordetella parapertussis
was developed with the insertion sequences IS
481
(
B. pertussis
) and IS
1001
(
B. parapertussis
) and evaluated with specimens from 520 consecutive patients presenting with possible pertussis. No culture-positive–PCR-negative results occurred, giving the method a sensitivity of 100%. For
B. pertussis
, 58 of 520 patients (11.2%) were positive by PCR compared to 17 of 520 patients positive (3.3%) by culture. For
B. parapertussis
, 7 of 520 patients (1.3%) were positive by PCR compared to 2 of 520 patients positive (0.4%) by culture. Two patients were positive for both
B. pertussis
and
B. parapertussis
. Patient records were reviewed to determine the validity of PCR-positive–culture-negative results. Forty-two of 49 patients who could be evaluated fulfilled the criteria for a case definition of pertussis, with 32 patients being <1 year of age and having classical pertussis symptoms. The seven patients who did not fulfil the criteria were aged 7 to 55 years and had a persistent cough for >2 weeks. The method was also used to investigate a classroom outbreak in which
B. pertussis
culture was positive for 5 of 28 patients. All five culture-positive specimens were confirmed by PCR, and an additional eight were positive by PCR. Of 25 patients from a suspected pertussis outbreak in a girls’ dormitory, seven of seven specimens were negative for
B. pertussis
, although 13 of 25 patients were positive for
B. pertussis
immunoglobulin M (IgM) (2 of which produced equivocal IgA results, with 23 of 25 patients being negative). Five symptomatic patients were subsequently found to be positive (by IgM and particle agglutination assays) for
Mycoplasma pneumoniae
, demonstrating the value of PCR in rapidly excluding
B. pertussis
infection in an outbreak situation. Twenty-two of 71 (30.1%) throat swabs were positive by PCR compared to 2 of 71 (2.8%) throat swabs positive by culture, indicating that a reassessment of the use of throat swabs should be considered, particularly for older patients, in contact tracing, and in situations in which specimen collection is difficult.
Improved Silica-Guanidiniumthiocyanate DNA Isolation Procedure Based on Selective Binding of Bovine Alpha-Casein to Silica Particles
Authors: Boom René , Sol Cees , Beld Marcel , Weel Jan , Goudsmit Jaap , Wertheim-van Dillen Pauline
Published: December 2019

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.
PCR Detection of DNA Specific for
Trichosporon
Species in Serum of Patients with Disseminated Trichosporonosis
Authors: Nagai Hiroyuki , Yamakami Yuriko , Hashimoto Atsuro , Tokimatsu Issei , Nasu Masaru
Published: December 2019

Deep-seated trichosporonosis is a lethal opportunistic infection that disseminates rapidly and widely in immunocompromised patients, and early diagnosis is crucial for the treatment of this infection. We developed a novel nested-PCR assay that detects DNA specific for clinically important strains of
Trichosporon
in serum samples from patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26S rRNA genes of
Trichosporon asahii
. The specific fragment was amplified from
T. asahii
and
T. mucoides
, but not from other microorganisms, including some other basidiomycetous fungi (Cryptococcus,
Malassezia
,
Rhodotorula
, and
Sporobolomyces
). Target DNA was detected by the nested PCR with as little as 5 fg of the extracted DNA of
T. asahii
. In a study using 11 clinical samples, the specific fragment was detected by the nested PCR in 64% (7 of 11) of sera from patients with histologically diagnosed disseminated trichosporonosis, while glucuronoxylomannan antigen was detected in only 54% (6 of 11) of the samples. Our new nested-PCR assay using serum samples can be performed repeatedly throughout the course of the disease. In addition, not only can it be used for early diagnosis of trichosporonosis, but it may also be beneficial for monitoring its progress or response to therapy.
Genetic Multilocus Studies of Different Strains of
Cryptococcus neoformans
: Taxonomy and Genetic Structure
Authors: Bertout S. , Renaud F. , Swinne D. , Mallié M. , Bastide J.-M.
Published: December 2019

The genotypes of 107 strains of
Cryptococcus
isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of
C. neoformans
and
C. laurentii
were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.
Cholera in Vietnam: Changes in Genotypes and Emergence of Class I Integrons Containing Aminoglycoside Resistance Gene Cassettes in
Vibrio cholerae
O1 Strains Isolated from 1979 to 1996
Authors: Dalsgaard A. , Forslund A. , Tam N. V. , Vinh D. X. , Cam P. D.
Published: December 2019

The number of cholera cases and the mortality rates reported from different regions of Vietnam varied considerably in the period from 1979 to 1996, with between 2,500 and 6,000 cases reported annually from 1992 to 1995. Annual mortality rates ranged from 2.0 to 9.6% from 1979 to 1983 to less than 1.8% after 1983. Major cholera outbreaks were reported from the High Plateau region for the first time in 1994 and 1995; this is an area with limited access to health services and safe drinking-water supplies. All cases were associated with
Vibrio cholerae
O1. Using ribotyping, cholera toxin (CT) genotyping, and characterization of antibiotic susceptibility patterns and antibiotic resistance genes by PCR, we show that strains isolated after 1990 were clearly different from strains isolated before 1991. In contrast to strains isolated before 1991, 94% of 104 strains isolated after 1990 showed an identical ribotype R1, were resistant to sulfamethoxazole and streptomycin, and showed a different CT genotype. Furthermore, PCR analysis revealed that sulfamethoxazole-resistant strains harbored class I integrons containing a gene cassette
ant(3")-1a
encoding resistance to streptomycin and spectinomycin. This is, to our knowledge, the first report of class I integrons in
V. cholerae
. The development of cholera and the changes in the phenotypic and genotypic properties of
V. cholerae
O1 shown in the present study highlight the importance of monitoring
V. cholerae
O1 in Vietnam as in other parts of the world. In particular, the emergence of the new ribotype R1 strain containing class I integrons should be further studied.
Impact of Microbiology Practice on Cumulative Prevalence of Respiratory Tract Bacteria in Patients with Cystic Fibrosis
Authors: Shreve Michael R. , Butler Steven , Kaplowitz Haley J. , Rabin Harvey R. , Stokes Dennis , Light Michael , Regelmann Warren E. ,   Investigators for the Epidemiologic Study of Cystic Fibrosis
Published: December 2019

Investigators participating in the Epidemiologic Study of Cystic Fibrosis project began to collect microbiological, pulmonary, and nutritional data on cystic fibrosis (CF) patients at 180 North American sites in 1994. Part of this study was a survey undertaken in August 1995 to determine microbiology laboratory practices with regard to pulmonary specimens from CF patients. The survey included a section on test ordering, completed by a site clinician, and a section on test performance and reporting, completed by each site’s clinical microbiology laboratory staff. Seventy-nine percent of the surveys were returned. There was intersite consistency of microbiology laboratory practices in most cases. The majority of sites follow most of the CF Foundation consensus conference recommendations. There were differences in the frequency at which specimens for culture were obtained, in the use of selective media for
Staphylococcus aureus
and
Haemophilus influenzae
, and in the use of a prolonged incubation for
Burkholderia cepacia
. These variations in practice contribute to prevalence differences among sites and may result in differences in clinical care.
Comparison of CHROMagar Salmonella Medium and Hektoen Enteric Agar for Isolation of Salmonellae from Stool Samples
Authors: Gaillot Olivier , Di Camillo Patrick , Berche Patrick , Courcol René , Savage Colette
Published: December 2019

CHROMagar Salmonella (CAS), a new chromogenic medium, was retrospectively compared to Hektoen enteric agar (HEA) with 501
Salmonella
stock isolates and was then prospectively compared to HEA for the detection and presumptive identification of
Salmonella
spp. with 508 stool samples before and after enrichment. All stock cultures (100%), including cultures of H
2
S-negative isolates, yielded typical mauve colonies on CAS, while 497 (99%) isolates produced typical lactose-negative, black-centered colonies on HEA. Following overnight incubation at 37°C, a total of 20
Salmonella
strains were isolated from the 508 clinical samples. Sensitivities for primary plating and after enrichment were 95% (19 isolates) and 100% (20 isolates), respectively, for CAS and 80% (16 isolates) and 100% (20 isolates), respectively, for HEA. The specificity of CAS (88.9%) was significantly higher than that of HEA (78.5%;
P
< 0.0001). On the basis of its good sensitivity and specificity, CAS medium can be recommended for use for primary plating when human stool samples are screened for
Salmonella
spp.
ABC Medium, a New Chromogenic Agar for Selective Isolation of
Salmonella
spp
Authors: Perry John D. , Ford Michael , Taylor Jeffrey , Jones Amanda L. , Freeman Roger , Gould Frances K.
Published: December 2019

We describe a new chromogenic agar medium, ABC medium (αβ-chromogenic medium), which includes two substrates, 3,4-cyclohexenoesculetin-β-
d
-galactoside and 5-bromo-4-chloro-3-indolyl-α-
d
-galactopyranoside, to facilitate the selective isolation of
Salmonella
spp. This medium exploits the fact that
Salmonella
spp. may be distinguished from other members of the family
Enterobacteriaceae
by the presence of α-galactosidase activity in the absence of β-galactosidase activity. A total of 1,022 strains of
Salmonella
spp. and 300 other gram-negative strains were inoculated onto this medium. Of these, 1,019 (99.7%) strains of
Salmonella
spp. produced a characteristic green colony, whereas only 1 strain (0.33%) of non-
Salmonella
produced a green colony. A total of 283 stool samples were cultured onto desoxycholate citrate (DC) agar and ABC medium by direct inoculation and after selective enrichment in selenite broth. Overall, the sensitivity and specificity were superior for ABC medium (100 and 90.5%, respectively) than for DC agar (88 and 26.9%, respectively). We conclude that ABC medium offers a high degree of specificity for the detection of
Salmonella
spp. in stool samples.
Comparison of Five PCR Methods for Detection of
Helicobacter pylori
DNA in Gastric Tissues
Authors: Lu Jang-Jih , Perng Cherng-Lih , Shyu Rong-Yaun , Chen Chi-Hsiang , Lou Qinyuan , Chong Sonny K. F. , Lee Chao-Hung
Published: December 2019

Five different PCR methods for the detection of
Helicobacter pylori
were evaluated. The results of this study indicate that of the five PCR methods examined, the
ureC
(
glmM
) gene PCR is the most sensitive and specific for the detection of
H. pylori
in gastric biopsy specimens.
Streptococcus bovis
Clone Causing Two Episodes of Endocarditis 8 Years Apart
Authors: Mühlemann Kathrin , Graf Susanne , Täuber Martin G.
Published: December 2019

A patient had endocarditis caused by
Streptococcus bovis
twice 8 years apart. According to pulsed-field gel electrophoresis (PFGE) the two isolates were identical. Seven unrelated blood isolates of
S. bovis
yielded unique PFGE patterns. Considering this heterogeneous population structure our findings demonstrate the long-term persistence of an
S. bovis
clone in a patient with recurrent endocarditis.
In Vitro Susceptibilities of
Candida dubliniensis
Isolates Tested against the New Triazole and Echinocandin Antifungal Agents
Authors: Pfaller M. A. , Messer S. A. , Gee S. , Joly S. , Pujol C. , Sullivan D. J. , Coleman D. C. , Soll D. R.
Published: December 2019

Candida dubliniensis
is a newly recognized fungal pathogen causing mucosal disease in AIDS patients. Although preliminary studies indicate that most strains of
C. dubliniensis
are susceptible to established antifungal agents, fluconazole-resistant strains have been detected. Furthermore, fluconazole-resistant strains are easily derived in vitro, and these strains exhibit increased expression of multidrug resistance transporters, especially MDR1. Because of the potential for the development of resistant strains of
C. dubliniensis
, it is prudent to explore the in vitro activities of several of the newer triazole and echinocandin antifungals against isolates of
C. dubliniensis
. In this study we tested 71 isolates of
C. dubliniensis
against the triazoles BMS-207147, Sch 56592, and voriconazole and a representative of the echinocandin class of antifungal agents, MK-0991. We compared the activities of these agents with those of the established antifungal agents fluconazole, itraconazole, amphotericin B, and 5-fluorocytosine (5FC) by using National Committee for Clinical Laboratory Standards microdilution reference methods. Our findings indicate that the vast majority of clinical isolates of
C. dubliniensis
are highly susceptible to both new and established antifungal agents. Strains with decreased susceptibilities to fluconazole remained susceptible to the investigational agents as well as to amphotericin B and 5FC. The increased potencies of the new triazole and echinocandin antifungal agents may provide effective therapeutic options for the treatment of infections due to
C. dubliniensis.
Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of
Mycoplasma pneumoniae
Authors: Duffy Michael F. , Whithear Kevin G. , Noormohammadi Amir H. , Markham Philip F. , Catton Michael , Leydon Jennie , Browning Glenn F.
Published: December 2019

Serology remains the method of choice for laboratory diagnosis of
Mycoplasma pneumoniae
infection. Currently available serological tests employ complex cellular fractions of
M. pneumoniae
as antigen. To improve the specificity of
M. pneumoniae
diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned
M. pneumoniae
gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of
M. pneumoniae
could provide a species-specific diagnostic tool, although further assessment is required.
Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay
Authors: von Messling Veronika , Harder Timm C. , Moennig Volker , Rautenberg Peter , Nolte Ingo , Haas Ludwig
Published: December 2019

Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in
Heliothis virescens
larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND
50
) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND
50
titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.
Characterization of a Culturable “
Gastrospirillum hominis
” (
Helicobacter heilmannii
) Strain Isolated from Human Gastric Mucosa
Authors: Andersen L. P. , Boye K. , Blom J. , Holck S. , Nørgaard A. , Elsborg L.
Published: December 2019

Spiral organisms were isolated from an antral gastric mucosal biopsy specimen from a dyspeptic patient with gastritis. Only corkscrew-shaped organisms resembling “
Gastrospirillum hominis
” (“
Helicobacter heilmannii
”) but no
Helicobacter pylori
-like organisms were seen in histological sections.
H. pylori
was not cultured from specimens from this patient. On the basis of biochemical reactions, morphology, ultrastructure, and 16S DNA sequencing, the isolated “
G. hominis
” was shown to be a true
Helicobacter
sp. very similar to
Helicobacter felis
and the “
Gastrospirillum
” but was separate from
H. pylori
. “
G. hominis
” is a pleomorphic gram-negative cork-screw-shaped, motile rod with 3 to 8 coils and a wavelength of about 1 μm. In contrast to
H. pylori
, it has up to 14 sheathed flagellar uni- or bipolar fibrils but no periplasmic fibrils. “
G. hominis
” grows under microaerobic conditions at 36 and 41°C on 7% lysed, defibrinated horse blood agar plates within 3 to 7 days and can be subcultured under microaerobic but not under anaerobic conditions on media similar to those used for
H. pylori
and
H. felis
. The small translucent colonies were, in contrast to those of
H. felis
, indistinguishable from those of
H. pylori
. “
G. hominis
” is, like
H. pylori
and
H. felis
, motile, is oxidase, catalase, nitrite, nitrate, and urease positive, and produces alkaline phosphatase and arginine arylamidase. Like
H. pylori
and
H. felis
, it is sensitive to cephalothin (30-μg disc), resistant to nalidixic acid (30-μg disc), and sensitive to most other antibiotics. The 16S DNA sequence clusters “
G. hominis
” together with “
Gastrospirillum
,”
H. felis
,
Helicobacter bizzozeronii
,
Helicobacter salmonii, Helicobacter nemestrinae
,
Helicobacter acinonychis
, and
H. pylori
.
DNA Banding Pattern Polymorphism in Vancomycin-Resistant
Enterococcus faecium
and Criteria for Defining Strains
Authors: Morrison D. , Woodford N. , Barrett S. P. , Sisson P. , Cookson B. D.
Published: December 2019

The degree of DNA banding pattern polymorphism exhibited by vancomycin-resistant
Enterococcus faecium
(VREM) strains isolated on a renal unit over an 11-month period was investigated. Thirty VREM strains from different patients were analyzed by pulsed-field gel electrophoresis (PFGE; with extended run and optimal pulse times), ribotyping, plasmid profile analysis, biotyping, pyrolysis mass spectrometry, and antibiogram analysis. PFGE resolved 17 banding patterns which formed four distinct clusters at the 82% similarity level. Intercluster band differences ranged from 14 to 31 bands. The strains in one cluster, which contained seven patterns that differed from each other by one to seven bands and from the common pattern by five bands, were confirmed to be a single strain by four of the five other typing methods. The strains in a second cluster with eight patterns, which differed from each other by 1 to 12 bands, contained two subclusters. This subdivision was supported by ribotyping and biotyping. However, it was unclear whether these subclusters represented distinct strains. In one strain, marked polymorphism (patterns that differed from each other by up to four bands) was observed in the ribotype pattern. This study demonstrates the high degree of DNA banding pattern polymorphism found for some strains of VREM and illustrates the complexity involved in defining such strains.
Allelic Diversity of the
Helicobacter pylori
Vacuolating Cytotoxin Gene in South Africa: Rarity of the
vacA
s1a Genotype and Natural Occurrence of an s2/m1 Allele
Authors: Letley D. P. , Lastovica A. , Louw J. A. , Hawkey C. J. , Atherton J. C.
Published: December 2019

We describe the rarity of
Helicobacter pylori
strains of vacuolating cytotoxin type s1a (the type most commonly associated with peptic ulceration in the United States) among black and mixed-race South Africans. We also provide the first description of a naturally occurring strain with the
vacA
allelic structure s2/m1.
Comparison of the MB/BacT and BACTEC 460 TB Systems for Recovery of Mycobacteria from Various Clinical Specimens
Authors: Brunello Francesca , Favari Flavio , Fontana Roberta
Published: December 2019

A total of 1,830 specimens (75.7% respiratory and 24.3% nonrespiratory) were cultured in parallel with the MB/BacT and BACTEC 460 TB systems and on Lowenstein-Jensen (LJ) medium. Mycobacteria were identified from 173 (6.5%) specimens. The most common species recovered were
Mycobacterium tuberculosis
complex (65.9%),
Mycobacterium avium
complex (22.5%), and
Mycobacterium chelonae
(9.2%). The recovery rates by individual systems were 96.5, 99.4, and 95.9% for MB/BacT, BACTEC 460 TB, and LJ medium, respectively, for all mycobacteria; the recovery rates were 99.1, 100, and 98.2%, respectively, for
M. tuberculosis
complex alone. The difference among the recovery rates for all mycobacteria and those for individual species was not significant. The BACTEC 460 TB system detected
M. tuberculosis
isolates more rapidly than the MB/BacT system (8 versus 11.8 days for smear-positive specimens [
P
< 0.01] and 18 versus 21 days for smear-negative specimens [
P
< 0.05]), whereas the MB/BacT system more rapidly detected the nontuberculous mycobacteria (17.1 versus 12.7 days [
P
< 0.01]). These results indicate that the nonradiometric MB/BacT system is a rapid, sensitive, and efficient method for the recovery of
M. tuberculosis
and nontuberculous mycobacteria from both pulmonary and extrapulmonary clinical specimens.
Extremely High Incidence of Macrolide and Trimethoprim-Sulfamethoxazole Resistance among Clinical Isolates of
Streptococcus pneumoniae
in Taiwan
Authors: Hsueh Po-Ren , Teng Lee-Jene , Lee Li-Na , Yang Pan-Chyr , Ho Shen-Wu , Luh Kwen-Tay
Published: December 2019

From January 1996 to December 1997, 200 isolates of
Streptococcus pneumoniae
recovered from 200 patients treated at National Taiwan University Hospital were serotyped and their susceptibilities to 16 antimicrobial agents were determined by the agar dilution method. Sixty-one percent of the isolates were nonsusceptible to penicillin, exhibiting either intermediate resistance (28%) or high-level resistance (33%). About two-fifths of the isolates displayed intermediate or high-level resistance to cefotaxime, ceftriaxone, cefepime, imipenem, and meropenem. Extremely high proportions of the isolates were resistant to erythromycin (82%), clarithromycin (90%), and trimethoprim-sulfamethoxazole (TMP-SMZ) (87%). Among the isolates nonsusceptible to penicillin, 23.8% were resistant to imipenem; more than 60% displayed resistance to cefotaxime, ceftriaxone, cefepime, and carbapenems; 96.7% were resistant to erythromycin; and 100% were resistant to TMP-SMZ. All isolates were susceptible to rifampin and vancomycin. The MICs at which 50% and 90% of the isolates were inhibited were 0.12 and 1 μg/ml, respectively, for cefpirome, and 0.12 and 0.25 μg/ml, respectively, for moxifloxacin. Six serogroups or serotypes (23F, 19F, 6B, 14, 3, and 9) accounted for 77.5% of all isolates. Overall, 92.5% of the isolates were included in the serogroups or serotypes represented in the 23-valent pneumococcal vaccine. The incidence of macrolide and TMP-SMZ resistance for
S. pneumoniae
isolates in Taiwan in this study is among the highest in the world published to date.
Species Identification and Strain Differentiation of Dermatophyte Fungi by Analysis of Ribosomal-DNA Intergenic Spacer Regions
Authors: Jackson Colin J. , Barton Richard C. , Evans E. Glyn V.
Published: December 2019

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus
Trichophyton rubrum
. The polymorphisms were detected by hybridization of
Eco
RI-digested
T. rubrum
genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of
T. rubrum
. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease
Mva
I produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as
T. rubrum-T. soudanense
and
T. quinkeanum-T. schoenlenii
could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of
T. rubrum
and for species identification of common dermatophyte fungi.