bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitochondrial Cytochrome
b
Gene Analysis of
Aspergillus fumigatus
and Related Species
Authors: Wang Li , Yokoyama Koji , Miyaji Makoto , Nishimura Kazuko 
Published: December 2019

ABSTRACT

Nucleotide sequences of 426 bp from the mitochondrial (mt) cytochrome
b
genes of six anamorph species and two species of
Neosartorya
teleomophs of
Aspergillus
section
Fumigati
were determined. These sequences were used to build nucleotide- and amino acid-based trees for phylogenetic analysis. Thirteen strains of
A. fumigatus
including 10 clinical isolates of
A. fumigatus
, 1 type culture of
A. fumigatus
var.
fumigatus
, 1 type culture of
A. fumigatus
var.
ellipticus
, and 1 strain of
A. fumigatus
var.
albus
, had the same nucleotide sequences. One strain of
A. fumisynnematus
, two strains labeled
A. neoellipticus
, two strains of
A. viridinutans
, and one strain of
A. duricaulis
had distinct nucleotide and amino acid sequences. Two strains of
A. brevipes
were divided into two types. One produced a 1,500-bp fragment that included an intron. The nucleotide sequences of its two exons were similar to those of the
A. fumigatus
, and the derived amino acid sequence was the same as that for
A. fumigatus
. The other produced a 426-bp fragment and had the same nucleotide and amino acid sequences as
A. unilateralis. Neosartorya fischeri
var.
fischeri
and
N. stramenia
had nucleotide sequences that differed from that of
A. fumigatus
. These species possessed their own characteristic nucleotide sequences that differed from each other. In comparisons of homologous sequences from four other pathogenic species of
Aspergillus
, regions specific to section
Fumigati
were found. The mt cytochrome
b
gene analysis was valuable for the identification, classification, and phylogenetic analysis of isolates of section
Fumigati
.
Comparison of the Nucleotide Sequences of 16S rRNA, 444Ep-ank, andgroESLHeat Shock Operon Genes in Naturally OccurringEhrlichia equiand Human Granulocytic Ehrlichiosis Agent Isolates from Northern California
Authors: Chae Joon-seok , Foley Janet E. , Dumler J. Stephen , Madigan John E. 
Published: December 2019

ABSTRACTWe examined 11 naturally occurring isolates ofEhrlichia equiin horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes.Ehrlichia equiisolates were from Sierra (n= 6), Mendocino (n= 3), Sonoma (n= 1), and Marin (n= 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444Ep-ankandgroESLheat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444Ep-ankgene of the HGE agent andE. equiisolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of thegroESLheat shock operon gene fragment is identical amongE. equi, the HGE agent, andE. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent andE. equiisolates from northern California. These results suggest thatE. equiand the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.
Diagnosis and Differentiation ofMycoplasma hyopneumoniaeandMycoplasma hyorhinisInfections in Pigs by PCR Amplification of the p36 and p46 Genes
Authors: Caron J. , Ouardani M. , Dea S. 
Published: December 2019

ABSTRACTThe genome ofMycoplasma hyopneumoniaeencodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions withM. flocculareandM. hyorhinisreduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific forM. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with anM. hyopneumoniaeinfection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection withM. hyorhiniswas diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection ofM. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.
Development of a Highly Specific RecombinantToxocara canisSecond-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis
Authors: Yamasaki Hiroshi , Araki Kunioki , Lim Patricia Kim Chooi , Zasmy Ngah , Mak Joon Wah , Taib Radzan , Aoki Takashi 
Published: December 2019

ABSTRACTThe specificity of the recombinantToxocara canisantigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen fromT. canissecond-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 μg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis withParagonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected withAscarisand hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected withAscarisand hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods.
Assessment of Morphology for Rapid Presumptive Identification of
Mycobacterium tuberculosis
and
Mycobacterium kansasii
Authors: Attorri Silvia , Dunbar Sherry , Clarridge Jill E. 
Published: December 2019

ABSTRACT

Mycobacterium tuberculosis
often exhibits serpentine cording when grown in liquid medium, whereas
Mycobacterium kansasii
can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for
M. tuberculosis
(64%) and cross-barring was recognized within 63 of 76 (83%) of the specimens positive for
M. kansasii
, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for
M. tuberculosis
and 83, 95, 59, and 98%, respectively, for
M. kansasii
. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of
Mycobacterium avium
complex from
M. tuberculosis
and the long beaded forms of
Mycobacterium gordonae
from
M. kansasii. Mycobacterium asiaticum
and
Mycobacterium szulgai
, which rarely occur, are genetically related to
M. kansasii
and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of
M. tuberculosis
and
M. kansasii
, respectively, and as guides for initial probe selection to reduce costs.
A Multisite Trial Comparing Two Cytomegalovirus (CMV) pp65 Antigenemia Test Kits, Biotest CMV Brite and Bartels/Argene CMV Antigenemia
Authors: St. George Kirsten , Boyd Molly J. , Lipson Steven M. , Ferguson David , Cartmell G. Frank , Falk Leon H. , Rinaldo Charles R. , Landry Marie L. 
Published: December 2019

ABSTRACT
A total of 513 blood specimens, predominantly from organ transplant recipients, human immunodeficiency virus-positive patients, and bone marrow transplant recipients, were tested for cytomegalovirus (CMV) by culture and pp65 antigenemia across four test sites. Peripheral blood leukocytes were examined by using both the Biotest CMV Brite and the Bartels/Argene CMV Antigenemia kits. A total of 109 specimens were positive for CMV, 106 (97%) were positive by antigenemia, and 34 (31%) were positive by culture. According to the manufacturers' instructions, 150,000 cells were applied per slide for the Biotest kit and 200,000 cells per slide for the Bartels kit. A total of 93 specimens (88%) were positive by the Biotest kit, and 86 (81%) were positive by the Bartels kit. In specimens found to be positive by only one kit, the positive cell counts were low (median, 1; range, 1 to 7). When the data from all four sites were combined and analyzed, there was no statistical difference between the performance of the two kits; the Biotest and Bartels kits were found to be equivalent in sensitivity, specificity, and positive and negative predictive values for the detection of CMV pp65 antigenemia.
Quantitative Galactomannan Detection Is Superior to PCR in Diagnosing and Monitoring Invasive Pulmonary Aspergillosis in an Experimental Rat Model
Authors: Becker Martin J. , de Marie Siem , Willemse Diana , Verbrugh Henri A. , Bakker-Woudenberg Irma A. J. M. 
Published: December 2019

ABSTRACT

Two diagnostic tests, an
Aspergillus
-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (
P
< 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis.
New Agar Medium for Testing Susceptibility of
Mycobacterium tuberculosis
to Pyrazinamide
Authors: Heifets Leonid , Sanchez Tracy 
Published: December 2019

ABSTRACT

A new agar medium to perform pyrazinamide (PZA) susceptibility testing with
Mycobacterium tuberculosis
has been developed. This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it also differs from conventional Middlebrook 7H10/7H11 agar in that animal serum (fetal or calf bovine or fetal equine serum) is used instead of oleic acid-albumin-dextrose-catalase to support good growth of
M. tuberculosis
at the low pH of 6.0. A critical concentration of 900 or 1,200 μg of PZA/ml in this medium made it possible to differentiate between PZA-susceptible and PZA-resistant clinical isolates. This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple and inexpensive. In addition, it has the potential of being used for a direct susceptibility test with PZA, but this approach will require further confirmation. Further studies to develop critical concentrations of other drugs for this low-pH medium, as well as to investigate the possibility of cultivation in regular (non-CO
2
) incubators, are in progress.
Development of Reverse Transcription-PCR Assays Specific for Detection of Equine Encephalitis Viruses
Authors: Linssen Bettina , Kinney Richard M. , Aguilar Patricia , Russell Kevin L. , Watts Douglas M. , Kaaden Oskar-Rüger , Pfeffer Martin 
Published: December 2019

ABSTRACT
Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR–seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR–seminested PCR.
Detection of the Agent of Heartwater,
Cowdria ruminantium
, in
Amblyomma
Ticks by PCR: Validation and Application of the Assay to Field Ticks
Authors: Peter Trevor F. , Barbet Anthony F. , Alleman Arthur R. , Simbi Bigboy H. , Burridge Michael J. , Mahan Suman M. 
Published: December 2019

ABSTRACT

We have previously reported that the pCS20 PCR detection assay for
Cowdria ruminantium
, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector
Amblyomma
ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of
C. ruminantium
originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of
Ehrlichia chaffeensis
, which is closely related to
C. ruminantium
. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 10
7
to 10
4
organisms but dropped to 61 and 28%, respectively, with ticks bearing 10
3
and 10
2
organisms. The assay also detected
C. ruminantium
in collections of
Amblyomma hebraeum
and
Amblyomma variegatum
field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for
C. ruminantium
in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.
Evaluation of CHROMagar Staph. aureus, a New Chromogenic Medium, for Isolation and Presumptive Identification of
Staphylococcus aureus
from Human Clinical Specimens
Authors: Gaillot Olivier , Wetsch Muriel , Fortineau Nicolas , Berche Patrick 
Published: December 2019

ABSTRACT

CHROMagar Staph. aureus (CSA) is a new chromogenic medium for presumptive identification of
Staphylococcus aureus
as mauve colonies after 24 h of incubation. We conducted a preliminary study with 100
S. aureus
and 45 coagulase-negative
Staphylococcus
(CoNS) stock isolates plated on CSA. All
S. aureus
isolates yielded mauve colonies after 24 h of incubation at 37°C, while CoNS isolates grew as blue, white, or beige colonies. Culture on CSA was then prospectively compared to a conventional laboratory method, i.e., culture on 5% horse blood agar (HBA), catalase test, and latex agglutination test (HBA-catalase-latex), for isolation and presumptive identification of
S. aureus
from 2,000 consecutive clinical samples. Among the 310
S. aureus
isolates recovered by at least one of the two methods, 296 grew as typical mauve colonies on CSA, while only 254 yielded catalase-positive, latex-positive colonies on HBA. The sensitivity of CSA was significantly higher than that of the conventional method (95.5 and 81.9%, respectively;
P
< 0.001) and allowed the recovery of important clinical isolates that were undetected on blood agar. The specificities of the two methods were not significantly different, although that of CSA was slightly higher (99.4% versus 98.9% for HBA-catalase-latex;
P
= 0.08). On the basis of its excellent sensitivity and specificity, ease of identification of positive colonies, and absence of complementary testing, CSA can be recommended as a routine plating medium for presumptive identification of
S. aureus
in clinical specimens.
Comparison of Polyvinyl Alcohol Fixative with Three Less Hazardous Fixatives for Detection and Identification of Intestinal Parasites
Authors: Jensen B. , Kepley W. , Guarner J. , Anderson K. , Anderson D. , Clairmont J. , De l'aune William , Austin E. H. , Austin G. E. 
Published: December 2019

ABSTRACT

Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the “gold standard” for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS.
Blastocystis hominis
and
Iodamoeba bütschlii
were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example,
Entamoeba histolytica
(
Entamoeba dispar
) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise,
Chilomastix mesnili
was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while
Entamoeba coli
was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of
E. histolytica
,
E. coli
, and
C. mesnili
. Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA.
Comparison of a Commercial Enzyme-Linked Immunosorbent Assay with Immunofluorescence and Complement Fixation Tests for Detection of
Coxiella burnetii
(Q Fever) Immunoglobulin M
Authors: Field Peter R. , Mitchell Jody L. , Santiago Avelina , Dickeson David J. , Chan Sau-Wan , Ho David W. T. , Murphy Alan M. , Cuzzubbo Andrea J. , Devine Peter L. 
Published: December 2019

ABSTRACT

A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio
Coxiella burnetii
immunoglobulin M [IgM] ELISA, QFM-200) was compared to the indirect fluorescent antibody test (IFAT) for
C. burnetii
IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (
n
= 6), chlamydia (
n
= 5), or legionella (
n
= 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio
C. burnetii
(Q fever) IgM ELISA (
F
= 187) is superior to that of CFT (
F
= 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever.