bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of
Neisseria meningitidis
Strains Causing Disease in Complement-Deficient and Complement-Sufficient Patients
Authors: Fijen C. A. P. , Kuijper E. J. , Dankert J. , Daha M. R. , Caugant D. A.
Published: December 2019

Serotyping and serosubtyping of meningococci showed no difference between isolates from 44 complement-deficient persons and from 50 complement-sufficient persons with meningococcal disease. Multilocus enzyme electrophoretic typing of the meningococci revealed 54 electrophoretic types that were equally distributed among isolates from complement-deficient and complement-sufficient patients. Analysis of strains isolated from eight complement-deficient persons with 11 recurrences of meningococcal disease showed that one strain was identical to the strain previously isolated from the same individual. Our results indicate that there are no differences between the clonal distributions of strains infecting complement-deficient and complement-sufficient patients. Most recurrences were infections caused by different strains.
Competitive PCR for Quantification of BM5d Proviral DNA in Mice with AIDS
Authors: Casabianca Anna , Vallanti Giuliana , Magnani Mauro
Published: December 2019

Murine AIDS in C57BL/6 mice is caused by a unique mixture of murine leukemia viruses. We report the use of a competitive PCR to detect and quantitate BM5d proviral DNA. This assay allowed discrimination among endogenous wild-type murine retroviruses and BM5d sequences. Furthermore, the method was subsequently used to evaluate the amount of BM5d in infected mice and in infected AZT (zidovudine)-treated mice, providing an effective way to quantitatively evaluate drug efficacy in the murine AIDS model.
Evaluation of the RapID CB Plus System for Identification of Coryneform Bacteria andListeriaspp
Authors: Funke Guido , Peters Katja , Aravena-Roman Max
Published: December 2019

In a stress test, the recently introduced RapID CB Plus system (Remel Inc. [formerly Innovative Diagnostic Systems], Norcross, Ga.) was challenged with a diverse set of gram-positive rods comprising 345 strains of coryneform bacteria and 33 strains ofListeriaspp. representing a total of 49 different taxa. Overall, within 4 h, the system correctly identified 80.9% of the strains on the species level and 12.2% of the strains on the genus level. Only 3.7% strains were misidentified, and for 3.2% of the strains no identification was provided. Difficulties with the system were mainly due to occasional uncertainties in reading reactions for acid production from carbohydrates and, to a lesser extent, aminopeptidase reactions. It is concluded that the system may also perform well under the conditions of a routine clinical laboratory.
Development of a PCR-Restriction Fragment Length Polymorphism Assay Using the Nucleotide Sequence of the
Helicobacter hepaticus
Urease Structural Genes
ureAB
Authors: Shen Zeli , Schauer David B. , Mobley Harry L. T. , Fox James G.
Published: December 2019

Infection with
Helicobacter hepaticus
causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters,
H. pylori
and
H. mustelae
,
H. hepaticus
possesses a high level of urease activity. However, the
H. hepaticus
urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from
H. hepaticus
genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial
H. hepaticus ureA
gene product was found to exhibit 60% identity and 75% similarity to the predicted
H. pylori
UreA. The deduced amino acid sequence of a partial
H. hepaticus ureB
gene product exhibited 75% identity and 87% similarity to the predicted
H. pylori
UreB. Diversity among
H. hepaticus
isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the
ureAB
open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease
Hha
I. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of
H. hepaticus
during an 18 month in vivo colonization study, suggesting that the urease genes of
H. hepaticus
are stable. The urease genes among
H. hepaticus
strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that
H. hepaticus
has urease structural genes which are homologous to those of the gastric
Helicobacter
species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.
Cat Scratch Disease: the Rare Role of
Afipia felis
Authors: Giladi Michael , Avidor Boaz , Kletter Yehudith , Abulafia Suzy , Slater Leonard N. , Welch David F. , Brenner Don J. , Steigerwalt Arnold G. , Whitney Anne M. , Ephros Moshe
Published: December 2019

Since its isolation in 1988,
Afipia felis
has been associated with cat scratch disease (CSD) in only one report and its role in CSD has been questioned. We have cultured
A. felis
from a lymph node of a patient with CSD. 16S rRNA gene sequencing, DNA relatedness studies, fatty acid analysis, and PCR of the
A. felis
ferredoxin gene showed that the isolate is identical to the previously reported
A. felis
isolate. To determine the role of
A. felis
in CSD, PCR of the 16S rRNA gene followed by hybridizations with specific probes were performed with lymph node specimens from CSD patients. All 32 specimens tested positive for
Bartonella henselae
and negative for
A. felis
. We conclude that
A. felis
is a rare cause of CSD. Diagnostic tests not conducive to the identification of
A. felis
might cause the diagnosis of CSD due to
A. felis
to be missed.
Chronic Prosthetic Hip Infection Caused by a Small-Colony Variant of
Escherichia coli
Authors: Roggenkamp Andreas , Sing Andreas , Hornef Mathias , Brunner Ulrich , Autenrieth Ingo B. , Heesemann Jürgen
Published: December 2019

From two different specimens of a chronic prosthetic hip infection taken at an interval of 2 months a slow-growing gram-negative bacterium was isolated in pure culture. The strain grew with the typical features of a small-colony variant (SCV). 16S rRNA sequencing identified the bacterium as
Escherichia coli
. Biochemical characterization demonstrated multiple phenotypic alterations of a mutant carrying a defect in the heme biosynthetic pathway (Hem
−
): (i) catalase and nitrate reductase reactions were both negative, (ii) a negative benzidine reaction demonstrated the lack of heme-containing cytochromes, and (iii) growth stimulation under anaerobic conditions as well as gentamicin resistance indicated defective aerobic respiration. PCR and Southern hybridization demonstrated that the mutation of the SCV of
E. coli
was localized in the
hemB
gene and was most likely due to a deletion of the
hemB
gene. On blood agar plates revertants were recognized growing as normal-sized colonies between the dominant small colonies of the strain. Feeding experiments indicated that the revertants but not the small colonies were permeable for hemin. A strong antibody response against the infecting SCV of
E. coli
was found. To our knowledge, this is the first report of a Hem
−
E. coli
strain as the etiological agent of a chronic bacterial infection.
Species Identification and Virulence Attributes of
Saccharomyces boulardii
(nom. inval.)
Authors: McCullough Michael J. , Clemons Karl V. , McCusker John H. , Stevens David A.
Published: December 2019

Saccharomyces boulardii
(nom. inval.) has been used for the treatment of several types of diarrhea. Recent studies have confirmed that
S. boulardii
is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment. The aim of the present study was to identify strains of
S. boulardii
to the species level and assess their virulence in established murine models. Three strains of
S. boulardii
were obtained from commercially available products in France and Italy. The three
S. boulardii
strains did not form spores upon repeated testing. Therefore, classical methods used for the identification of
Saccharomyces
spp. could not be undertaken. Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5.8S ribosomal DNA) showed that the three isolates of
S. boulardii
were not separable from authentic isolates of
Saccharomyces cerevisiae
with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of
Saccharomyces
could be differentiated. RFLP analysis of cellular DNA with
Eco
RI showed that all three strains of
S. boulardii
had identical patterns and were similar to other authentic
S. cerevisiae
isolates tested. Therefore, the commercial strains of
S. boulardii
available to us cannot be genotypically distinguished from
S. cerevisiae
. Two
S. boulardii
strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of
S. cerevisiae
. The results of the present study show that these
S. boulardii
strains are asporogenous strains of the species
S. cerevisiae
, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection. Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.
Clonal Distribution of Invasive
Neisseria meningitidis
Isolates from the Norwegian County of Telemark, 1987 to 1995
Authors: Aakre Randi Kersten , Jenkins Andrew , Kristiansen Bjørn-Erik , Frøholm L. Oddvar
Published: December 2019

Forty-two
Neisseria meningitidis
isolates were obtained from patients with meningococcal disease in the Norwegian county of Telemark (January 1987 to March 1995), and all were compared by PCR amplicon restriction endonuclease analysis (PCR-AREA) of the
dhps
gene, chromosomal DNA fingerprinting, and serological analysis. PCR-AREA divided the isolates into 11 classes, of which 4, comprising 15, 8, 6, and 2 isolates, were clonal while the remaining 8 classes were genetically heterogeneous or contained only 1 isolate. Three of the four clonal classes could be tentatively equated with recognized epidemic clones (ET5, ET37, and cluster A4) on the basis of their phenotypic characteristics, while the remaining clone appears to be new. There were significant differences in the geographical distribution of clones, with class 1 (ET5-like) isolates significantly overrepresented in rural parts of Telemark. Class 1 (ET5-like) isolates occurred throughout the study period and were dominant in 1987. Class 2 (ET37-like) isolates occurred from 1988 to 1992, and class 3 isolates (with no recognizable ET affinities) were found only in 1991 and 1992.
Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR
Authors: Harris Eva , Roberts T. Guy , Smith Leila , Selle John , Kramer Laura D. , Valle Sonia , Sandoval Erick , Balmaseda Angel
Published: December 2019

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.
Molecular Analysis by Pulsed-Field Gel Electrophoresis and Antibiogram of
Streptococcus pneumoniae
Serotype 6B Isolates from Selected Areas within the United States
Authors: Rudolph Karen M. , Parkinson Alan J. , Roberts Marilyn C.
Published: December 2019

Fifty-eight clinical isolates of
Streptococcus pneumoniae
serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases
Apa
I and
Sma
I were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The
Apa
I and
Sma
I PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related
Apa
I and
Sma
I PFGE patterns, differing by only one band after restriction with
Apa
I. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant
Apa
I and
Sma
I PFGE patterns seen in the Alaskan isolates; 5 others were highly related (±1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional
Apa
I plus
Sma
I PFGE patterns were observed. The 28 isolates from the various contiguous states had 22
Apa
I plus
Sma
I PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.
Detection of
Ehrlichia phagocytophila
DNA in
Ixodes ricinus
Ticks from Areas in Switzerland Where Tick-Borne Fever Is Endemic
Authors: Pusterla Nicola , Huder Jon B. , Lutz Hans , Braun Ueli
Published: December 2019

A total of 1,523 adult
Ixodes ricinus
ticks were collected from regions where bovine ehrlichiosis is endemic and were examined for
Ehrlichia phagocytophila
via PCR. Of the ticks from cattle with ehrlichiosis, the ticks from healthy cattle, and the free-living ticks, 26.5% (18 of 68), 4.4% (35 of 802), and 0.8% (5 of 653), respectively, were positive.
Novel Intestinal
Helicobacter
Species Isolated from Cotton-Top Tamarins (
Saguinus oedipus
) with Chronic Colitis
Authors: Saunders Kim E. , Shen Zeli , Dewhirst Floyd E. , Paster Bruce J. , Dangler Charles A. , Fox James G.
Published: December 2019

A disease similar to ulcerative colitis in humans has been identified in cotton-top tamarins (CTTs) in captivity. The clinical signs include weight loss, diarrhea, and rectal bleeding with the pathological features and biochemical abnormalities of ulcerative colitis. Approximately 25 to 40% of these animals develop colon cancer after 2 to 5 years of captivity. An infectious etiology has been proposed; however, no microbial agent to date has been identified.
Helicobacter
spp. have been associated with enterocolitis and inflammatory bowel disease (IBD) in humans and animals. Infection with
Helicobacter pylori
or
Helicobacter mustelae
is associated with an increased risk of gastric adenocarcinoma and lymphoma of the mucosa-associated lymphoid tissue.
Helicobacter hepaticus
causes hepatitis, hepatic adenomas, and hepatocellular carcinomas in susceptible strains of mice. The aim of this study was to assess a colony of CTTs with a high incidence of IBD and colon cancer for the presence of colonic
Helicobacter
spp. A fusiform, gram-negative bacterium with bipolar flagella and periplasmic fibers was isolated from the feces of CTTs. The bacterium grew under microaerobic conditions at 37 and 42°C but not at 25°C, did not hydrolyze urea, was positive for catalase and oxidase, did not reduce nitrate to nitrite, did not hydrolyze indoxyl acetate or alkaline phosphatase, and was resistant to nalidixic acid, cephalothin, and trimethoprim-sulfamethoxazole. On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel
Helicobacter
species. This is the first
Helicobacter
isolated from CTTs. Further studies are needed to elucidate the role of this novel
Helicobacter
sp. in the pathogenesis of ulcerative colitis and colonic adenocarcinoma in CTTs.
Semiquantitative Species-Specific Detection of
Bartonella henselae
and
Bartonella quintana
by PCR-Enzyme Immunoassay
Authors: Sander Anna , Penno Susanne
Published: December 2019

Bartonella henselae
is the main causative agent of cat-scratch disease, and both
B. henselae
and
Bartonella quintana
cause angioproliferative disorders such as bacillary angiomatosis. To increase the sensitivity of
Bartonella
detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA). The 16S rRNA gene was selected as the target sequence. Internal nucleotide sequences derived from the amplified 16S rRNA region were used to develop species-specific oligonucleotide probes for
B. henselae
and
B. quintana
. Biotin-labeled PCR products were immobilized on streptavidin-coated microtiter plates, hybridized to a digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase conjugate. No cross-hybridization with other
Bartonella
or non-
Bartonella
species was observed. This EIA was as sensitive as dot blot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels. Serial dilutions of
B. henselae
and
B. quintana
suspensions demonstrated that an optical density (OD) of approximately 0.200 was equivalent to 5 CFU in the reaction mixture. By comparing the OD of the bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10
3
to 10
6
CFU/ml. The PCR-EIA developed in the present study is a rapid, sensitive, and simple method for the diagnosis of
B. henselae
and
B. quintana
infections.
Epidemiological Evidence for Dormant
Cryptococcus neoformans
Infection
Authors: Garcia-Hermoso Dea , Janbon Guilhem , Dromer Françoise
Published: December 2019

To date, the time of acquisition of a
Cryptococcus neoformans
infectious strain has never been studied. We selected a primer, (GACA)
4
, and a probe, CNRE-1, that by randomly amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP), respectively, regrouped strains from control samples of
C. neoformans
var.
grubii
environmental isolates according to their geographical origins. The two typing techniques were then used to analyze 103 isolates from 29 patients diagnosed with cryptococcosis in France. Nine of the 29 patients lived in Africa a median of 110 months prior to moving to France; 17 of the patients originated from Europe. Results showed a statistically significant clustering of isolate subtypes from patients originating from Africa compared to those from Europe. We conclude that the patients had acquired the
C. neoformans
infectious strain long before their clinical diagnoses were made.
Usefulness of Seminested Multiplex PCR in Surveillance of Imported Malaria in Spain
Authors: Rubio J. M. , Benito A. , Berzosa P. J. , Roche J. , Puente S. , Subirats M. , López-Vélez R. , García L. , Alvar J.
Published: December 2019

The use of a new PCR-based method for the diagnosis of malaria in the Spanish Malaria Reference Laboratory has promoted an increase in confirmed cases of malaria. From August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients were received from the hospitals of the Spanish National Health System. Most of the patients came from west-central African countries (85%). This molecular method showed more sensitivity and specificity than microscopy, detecting 12.4% more positive samples than microscopy and 13% of mixed infections undetectable by Giemsa stain.
Plasmodium falciparum
was the main species detected, with 68% of the total positive malaria cases, followed by
Plasmodium malariae
(29%),
Plasmodium vivax
(14%), and
Plasmodium ovale
(7%), including mixed infections in all cases. This report consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a seminested multiplex PCR permitted confirmation of the origins of the infections and the
Plasmodium
species involved and confirmation of the effectiveness of drug treatments. This PCR also allowed the detection of the presence in Spain of primaquine-tolerant
P. vivax
strains from west-central Africa, as well as the detection of a
P. falciparum
infection induced by transfusion.
Prophage Carriage as a Molecular Epidemiological Marker in
Streptococcus pneumoniae
Authors: Severina Elena , Ramirez Mario , Tomasz Alexander
Published: December 2019

The great majority of clinical isolates of
Streptococcus pneumoniae
carry prophages that may be identified through their hybridization with a DNA probe specific for the pneumococcal
lytA
gene (M. Ramirez, E. Severina, and A. Tomasz, J. Bacteriol. 181:3618–3625, 1999). We now show that the
lytA
hybridization pattern of chromosomal
Sma
I digests is stable for a given strain during extensive serial culturing in the laboratory; the pattern is specific for the strain’s clonal type, as defined by pulsed-field gel electrophoretis (PFGE) pattern, and variations in PFGE subtypes may be explained by changes in the number and chromosomal localization of this prophage(s). These observations indicate that the
lytA
hybridization pattern may be used as a molecular epidemiological marker that offers additional resolution of the genetic background of
S. pneumoniae
strains.
Evaluation of a
vanA
-Specific PCR Assay for Detection of Vancomycin-Resistant
Enterococcus faecium
during a Hospital Outbreak
Authors: Roger Michel , Faucher Marie-Claude , Forest Pierre , St-Antoine Pierre , Coutlée François
Published: December 2019

We investigated the use of PCR as an alternative to culture of fecal samples for detection of
vanA
-containing
Enterococcus faecium
during a recent hospital outbreak. Rectal swabs collected consecutively from 223 patients were analyzed by culture with and without enrichment broth and by
vanA
-specific PCR of enrichment broth samples. Fifty-five specimens were positive for
vanA
-containing
E. faecium
by at least one method. The sensitivities of the
vanA
-specific PCR assay and agar culture with and without enrichment broth were 94.5, 98, and 89%, respectively. All three methods were 100% specific. Final results were obtained much more rapidly by PCR (within 24 to 30 h of specimen submission) than by the culture methods (4 to 5 days). Thus, PCR is an accurate and rapid alternative to culture for detection of vancomycin-resistant enterococci during hospital outbreaks.
Antibiotic Susceptibilities of Group C and Group G Streptococci Isolated from Patients with Invasive Infections: Evidence of Vancomycin Tolerance among Group G Serotypes
Authors: Zaoutis Theoklis , Schneider Barbara , Steele Moore Lynn , Klein Joel D.
Published: December 2019

A retrospective review of medical records for 32 patients with invasive group C streptococcus (GCS) or group G streptococcus (GGS) infections was performed. MICs and minimum bactericidal concentrations (MBCs) of penicillin, erythromycin, and vancomycin for all isolates were obtained. Tolerance of vancomycin, defined as an MBC 32 or more times higher than the MIC, was exhibited by 18 GGS isolates (54%). The identification of tolerance in clinical isolates of GGS and GCS may have clinical implications in treating these seriously ill patients.
Maxillary Sinusitis Caused by Medusoid Form of
Schizophyllum commune
Authors: Sigler Lynne , Bartley James R. , Parr Dinah H. , Morris Arthur J.
Published: December 2019

We present a case of maxillary sinusitis in a diabetic female caused by the basidiomycete fungus
Schizophyllum commune
. Identification of the isolate was hampered by its atypical features. Subcultures formed sterile medusoid structures from nonclamped mycelia until spontaneous dikaryotization resulted in the development of characteristic fan-shaped fruiting bodies. Identification was confirmed by the presence of spicules formed on the hyphae and by vegetative compatibility with known isolates.
Optimizing Culture of
Chlamydia pneumoniae
by Using Multiple Centrifugations
Authors: Pruckler Janet M. , Masse Nirupama , Stevens Valerie A. , Gang Liu , Yang Yonghong , Zell Elizabeth R. , Dowell Scott F. , Fields Barry S.
Published: December 2019

Three methods for the recovery of
Chlamydia pneumoniae
from spiked nasopharyngeal and blood specimens, including extended culture and additional centrifugations, were compared. Additional centrifugations and a 7-day culture time resulted in a 500- to 5,000-fold increase in the number of detectable inclusion-forming units.
Pneumocystis carinii
Carriage in Immunocompetent Patients with Primary Pulmonary Disorders as Detected by Single or Nested PCR
Authors: Sing Andreas , Roggenkamp Andreas , Autenrieth Ingo B. , Heesemann Jürgen
Published: December 2019

Ninety-five bronchoalveolar lavage specimens from 63 immunocompetent adult patients with primary pulmonary disease were analyzed for
Pneumocystis carinii
colonization by primary and nested PCR. Twelve of 63 patients (19%) were PCR positive. None of them developed
P. carinii
pneumonia. These results suggest that
P. carinii
carriage may exist in immunocompetent patients with underlying pulmonary disease.
Comparison of the Quantiplex Version 3.0 Assay and a Sensitized Amplicor Monitor Assay for Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma Samples
Authors: Highbarger Helene C. , Alvord W. Gregory , Jiang Min Kang , Shah Akram S. , Metcalf Julia A. , Lane H. Clifford , Dewar Robin L.
Published: December 2019

This study evaluated correlation and agreement between version 3 of the Quantiplex human immunodeficiency virus type 1 (HIV-1) RNA assay (v3 branched DNA [bDNA]) and a sensitized Amplicor HIV-1 Monitor assay (reverse transcription [RT]-PCR) for the measurement of HIV RNA. Three hundred eighteen samples from 59 randomly selected, HIV-1-seropositive persons on various drug protocols from the National Institute of Allergy and Infectious Diseases HIV outpatient clinic were studied. The results indicate that v3 bDNA and RT-PCR are highly correlated (
r
= 0.98) and are in good agreement (mean difference in log
10
copies/ml ± 2 standard deviations = 0.072 ± 0.371). The relationship between values obtained by both assays is given by the following equation: log
10
v3 bDNA = −0.0915 + 1.0052 · log
10
RT-PCR. This represents a 1.026-fold difference between log
10
RT-PCR values and log
10
v3 bDNA values.
Comparison of Variant-Specific Hybridization and Single-Strand Conformational Polymorphism Methods for Detection of Mixed Human Papillomavirus Type 16 Variant Infections
Authors: Emeny Rebecca T. , Herron John R. , Xi Long Fu , Koutsky Laura A. , Kiviat Nancy B. , Wheeler Cosette M.
Published: December 2019

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.
Identification of
Candida dubliniensis
Based on Temperature and Utilization of Xylose and α-Methyl-
d
-Glucoside as Determined with the API 20C AUX and Vitek YBC Systems
Authors: Gales A. C. , Pfaller M. A. , Houston A. K. , Joly S. , Sullivan D. J. , Coleman D. C. , Soll D. R.
Published: December 2019

To have a better understanding of the role of
Candida dubliniensis
in clinical infections, it is essential that microbiology laboratories can identify this species rapidly and accurately in clinical specimens.
C. dubliniensis
has been reported to lack the ability to utilize xylose (XYL) and α-methyl-
d
-glucoside (MDG) and to grow poorly or not at all at 45°C, whereas
Candida albicans
isolates utilize XYL and MDG and usually grow well at 45°C. We tested 66 isolates of
C. dubliniensis
and 100 isolates of
C. albicans
with both the API 20C AUX and Vitek YBC systems to evaluate the ability of the XYL and MDG tests contained within each of these systems to distinguish between the two species. The ability to grow at 45°C was also examined. None of the
C. dubliniensis
isolates grew at 45°C, and 23 of 100
C. albicans
isolates (23%) exhibited poor or no growth at 45°C. The XYL and MDG tests contained within the API 20C AUX system were both negative for all 66
C. dubliniensis
isolates and were positive for 98 (XYL) and 56 (MDG) of the 100
C. albicans
isolates. With the Vitek system, 64 of 66
C. dubliniensis
isolates (97.0%) were XYL negative and 63 (95.0%) were MDG negative. Conversely, 96 of 100
C. albicans
isolates (96.0%) were XYL positive and 100 (100.0%) were MDG positive with the Vitek system. Clinical microbiology laboratories could use lack of growth at 45°C and a negative XYL test with either the API 20C AUX or Vitek yeast identification system to provide a presumptive identification of
C. dubliniensis
. A negative MDG test result with either system would also be helpful but may misclassify
C. albicans
as
C. dubliniensis
, especially when the API 20C AUX system is used.
Adenoviruses from Human Immunodeficiency Virus-Infected Individuals, Including Two Strains That Represent New Candidate Serotypes Ad50 and Ad51 of Species B1 and D, Respectively
Authors: De Jong Jan C. , Wermenbol Anton G. , Verweij-Uijterwaal Marja W. , Slaterus Kees W. , Wertheim-Van Dillen Pauline , Van Doornum Gerard J. J. , Khoo Saye H. , Hierholzer John C.
Published: December 2019

Adenovirus (Ad) isolates from a large number of human immunodeficiency virus (HIV)-infected individuals were compared serologically and genetically with Ad isolates from immunocompetent patients. Between 1982 and 1994, stool and urine samples from 137 subjects with AIDS hospitalized in The Netherlands yielded 143 Ad strains. Forty additional Ad strains were obtained from 35 HIV-positive patients in Manchester, United Kingdom, in 1992 and 1993. Of these 183 HIV-associated Ad strains, 84% belonged to species D and 3% belonged to species C. These strains were compared with 2,301 Ad strains collected during general diagnostic examinations in The Netherlands from 1973 to 1992. Of the latter strains, 5% belonged to species D and 49% belonged to species C. Two of the Ads isolated from fecal specimens of AIDS patients represent new serotypes: candidate Ad serotype 50 (prototype strain, Wan) of subspecies B1 and candidate Ad serotype 51 (prototype strain, Bom) of species D. The DNA restriction enzyme patterns of strains Wan and Bom differed from the patterns of all established prototypes.
Porphyromonas gingivalis
Strain Variability and Periodontitis
Authors: Griffen Ann L. , Lyons Sharon R. , Becker Mitzi R. , Moeschberger Melvin L. , Leys Eugene J.
Published: December 2019

To determine if there is variability in virulence among strains of
Porphyromonas gingivalis
in human periodontitis, their distribution in a group of subjects with clear indicators of periodontitis and in a healthy, age-matched control group was examined. The presence of heteroduplex types of
P. gingivalis
in the two groups was determined with a PCR-based assay. This assay relied on detection of polymorphisms in the ribosomal internal spacer region (ISR). ISR fragments generated by PCR with
P. gingivalis
-specific primers were hybridized to fragments from reference strains, and the formation of heteroduplexes from the hybridization of nonidentical sequences was observed by polyacrylamide gel electrophoresis. Characteristic fingerprints from comparison with a panel of reference strains allowed the identification of heteroduplex types in clinical samples. One hundred thirty adults with periodontitis and 181 controls were sampled. With this approach, 11 heteroduplex types of
P. gingivalis
were detected in the population. Sufficient numbers were available for statistical analysis of six of these types. Heteroduplex type hW83 was found to be very strongly associated with periodontitis (
P
= 0.0000), and two additional types, h49417 and hHG1691, were also significantly associated with disease. The remaining types, h23A4, h381, and hA7A1, were detected more frequently in subjects with periodontitis than in healthy subjects, but the difference was not significant. These data indicate that virulence in human periodontitis varies among strains of
P. gingivalis
, and they identify an apparently highly virulent subgroup.
Detection and Reporting of Organisms Producing Extended-Spectrum β-Lactamases: Survey of Laboratories in Connecticut
Authors: Tenover Fred C. , Mohammed M. Jasmine , Gorton Timothy S. , Dembek Zygmunt F.
Published: December 2019

Extended-spectrum β-lactamases (ESBLs) are enzymes produced in some gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. They are most common in
Klebsiella
spp. and
Escherichia coli
but are present in a variety of
Enterobacteriaceae
. Resistance mediated by these enzymes can be difficult to detect depending on the antimicrobial agents tested. AmpC β-lactamases are related to the chromosomal enzymes of
Enterobacter
and
Citrobacter
spp. and also mediate resistance to extended-spectrum cephalosporins and aztreonam in addition to cephamycins, such as cefoxitin. Unlike ESBLs, however, AmpC β-lactamases are not inhibited by clavulanic acid or other similar compounds. To assess the abilities of various antimicrobial susceptibility testing methods to detect ESBLs, we sent three ESBL-producing organisms, one AmpC-producing organism, and a control strain that was susceptible to extended-spectrum cephalosporins to 38 laboratories in Connecticut for testing. Eight (21.0%) of 38 labs failed to detect extended-spectrum cephalosporin or aztreonam resistance in any of the ESBL- or AmpC-producing isolates. Errors were encountered with both automated and disk diffusion methods. Conversely, seven (18.4%) labs categorized at least some of the four resistant isolates as potential ESBL producers and reported the results with the extended-spectrum cephalosporins and aztreonam as resistant as suggested by current National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The percentage of laboratories that failed to detect resistance in the ESBL or AmpC isolates ranged from 23.7 to 31.6% depending on the type of enzyme present in the test organism. This survey suggests that many laboratories have difficulty detecting resistance in ESBL and AmpC-producing organisms and may be unaware of the NCCLS guidelines on modifying susceptibility testing reports for ESBL-producing strains.
Direct
mecA
Detection from Blood Culture Bottles by Branched-DNA Signal Amplification
Authors: Zheng X. , Kolbert C. P. , Varga-Delmore P. , Arruda J. , Lewis M. , Kolberg J. , Cockerill F. R. , Persing D. H.
Published: December 2019

A branched-DNA (bDNA) signal amplification method was used to detect the
mecA
gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the
mecA
gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the
mecA
gene by bDNA signal amplification is (i) sensitive enough to detect
mecA
directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.
Evaluation of AMPLILINK Software for the COBAS AMPLICOR System
Authors: Kessler Harald H. , Jungkind Donald , Stelzl Evelyn , Direnzo Sue , Vellimedu Srinivas K. , Pierer Karen , Santner Brigitte , Marth Egon
Published: December 2019

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.
Associations between Virulence Factors of Shiga Toxin-ProducingEscherichia coliand Disease in Humans
Authors: Boerlin Patrick , McEwen Scott A. , Boerlin-Petzold Franziska , Wilson Jeffrey B. , Johnson Roger P. , Gyles Carlton L.
Published: December 2019

Associations between known or putative virulence factors of Shiga toxin-producingEscherichia coliand disease in humans were investigated. Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx2) and isolates from serotypes reported in humans. Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome. The enterohemorrhagicE. coli(EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models. A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded. This result warrants further investigations of this topic. Multivariate analysis revealed an interaction between theeaeandstx2genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2. A strong statistical association was observed between thestx2gene and severity of disease for a set of 112 human isolates from eight major serotypes. A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.
Identification of the Enteropathogens
Campylobacter jejuni
and
Campylobacter coli
Based on the
cadF
Virulence Gene and Its Product
Authors: Konkel Michael E. , Gray Sean A. , Kim Bong J. , Garvis Steven G. , Yoon Julie
Published: December 2019

Campylobacter jejuni
and
Campylobacter coli
are common causes of gastroenteritis in humans. Infection with
C. jejuni
or
C. coli
is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for
C. jejuni
and
C. coli
isolates based on the
cadF
virulence gene and its product. The
cadF
gene from
C. jejuni
and
C. coli
encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%)
C. jejuni
isolates and 5 of 6 (83.3%)
C. coli
isolates with primers designed to amplify an internal fragment of the
cadF
gene. PCR was then used to amplify
Campylobacter
DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every
C. jejuni
and
C. coli
isolate tested, as judged by immunoblot analysis with a rabbit anti-
C. jejuni
37-kDa serum. In addition, methanol-fixed samples of whole-cell
C. jejuni
and
C. coli
were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the
cadF
gene and its product are conserved among
C. jejuni
and
C. coli
isolates and that a PCR assay based on the
cadF
gene may be useful for the detection of
Campylobacter
organisms in food products.
Rapid Identification and Typing ofStaphylococcus aureusby PCR-Restriction Fragment Length Polymorphism Analysis of thearoAGene
Authors: Marcos Javier Yugueros , Soriano Alberto Cascón , Salazar María Sánchez , Moral Carmen Hernanz , Ramos Susana Suárez , Smeltzer Mark S. , Carrasco German Naharro
Published: December 2019

TheStaphylococcus aureus aroAgene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from allS. aureusstrains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when otherStaphylococcusspp. or genera were analyzed by using the same pair of primers. The detection limit forS. aureuscells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 102CFU) whenS. aureuscells were suspended in sterilized whole milk.TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.
Rapid Detection of theChlamydiaceaeand Other Families in the OrderChlamydiales: Three PCR Tests
Authors: Everett Karin D. E. , Hornung Linda J. , Andersen Arthur A.
Published: December 2019

Few identification methods will rapidly or specifically detect all bacteria in the orderChlamydiales, familyChlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized theChlamydiaceae: a multiplex test targeting theompAgene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the orderChlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation.
Phylogenetic Relationships of Varieties and Geographical Groups of the Human Pathogenic Fungus
Histoplasma capsulatum
Darling
Authors: Kasuga Takao , Taylor John W. , White Thomas J.
Published: December 2019

The phylogeny of 46 geographically diverse
Histoplasma capsulatum
isolates representing the three varieties
capsulatum
,
duboisii
, and
farciminosum
was evaluated using partial DNA sequences of four protein coding genes. Parsimony and distance analysis of the separate genes were generally congruent and analysis of the combined data identified six clades: (i) class 1 North American
H. capsulatum
var.
capsulatum
, (ii) class 2 North American
H. capsulatum
var.
capsulatum
, (iii) Central American
H. capsulatum
var.
capsulatum
, (iv) South American
H. capsulatum
var.
capsulatum
group A, (v) South American
H. capsulatum
var.
capsulatum
group B, and (vi)
H. capsulatum
var.
duboisii
. Although the clades were generally well supported, the relationships among them were not resolved and the nearest outgroups (
Blastomyces
and
Paracoccidioides
) were too distant to unequivocally root the
H. capsulatum
tree.
H. capsulatum
var.
farciminosum
was found within the South American
H. capsulatum
var.
capsulatum
group A clade. With the exception of the South American
H. capsulatum
var.
capsulatum
group A clade, genetic distances within clades were an order of magnitude lower than those between clades, and each clade was supported by a number of shared derived nucleotide substitutions, leading to the conclusion that each clade was genetically isolated from the others. Under a phylogenetic species concept based on possession of multiple shared derived characters, as well as concordance of four gene genealogies,
H. capsulatum
could be considered to harbor six species instead of three varieties.