bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated Specific Capture of Hepatitis C Virus RNA with Probes and Paramagnetic Particle Separation
Authors: Miyachi Hayato , Masukawa Atsuko , Ohshima Toshio , Hirose Toru , Impraim Chaka , Ando Yasuhiko
Published: May 2021

We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10
3
or 10
5
copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.
Phenotypic and Molecular Typing of Nosocomial Methicillin-Resistant
Staphylococcus aureus
Strains Susceptible to Gentamicin Isolated in France from 1995 to 1997
Authors: Galdbart Jacques-Olivier , Morvan Anne , El Solh Nevine
Published: May 2021

Methicillin-resistant strains susceptible to gentamicin (Gm
s
MRSA) have emerged since 1993 in several French hospitals. To study whether particular clones have spread in various French cities and whether some clones are related to gentamicin-resistant (Gm
r
) MRSA strains, various methods (antibiotyping, phage typing, determination of
Sma
I macrorestriction patterns before and after hybridization with IS
256
transposase and
aacA-aphD
probes) were used to compare 62 Gm
s
MRSA strains isolated from 1995 to 1997 in nine cities and 15 Gm
r
MRSA strains. Eighteen major
Sma
I genotypes were identified, of which 11 included only Gm
s
MRSA strains and 5 included only Gm
r
MRSA strains. Each of the Gm
r
MRSA strains contained 6 to 13
Sma
I fragments hybridizing with the insertion sequence IS
256
, of which a single band also hybridized with the
aacA-aphD
gene. No such hybridizing sequences were detected in 60 of the 62 Gm
s
MRSA strains. Thus, the divergence between Gm
r
and Gm
s
MRSA strains is revealed, not only by their distributions in distinct
Sma
I genotypes but also by the differences in hybridization patterns. Two of the 62 Gm
s
MRSA strains had the uncommon feature of carrying several
Sma
I bands hybridizing with IS
256
, suggesting that they are possibly related to the Gm
r
MRSA strains grouped in the same
Sma
I genotype. Five of the 11
Sma
I genotypes including only Gm
s
MRSA strains contained strains from diverse cities, isolated during different years and with different antibiograms, suggesting that some clones have spread beyond their cities of origin and persisted.
Restriction Fragment Length Polymorphism Analysis of
Mycobacterium tuberculosis
Isolated from Countries in the Western Pacific Region
Authors: Park Young-Kil , Bai Gill-Han , Kim Sang-Jae
Published: May 2021

A total of 422
Mycobacterium tuberculosis
isolates from eight countries were subjected to IS
6110
and IS
1081
DNA fingerprinting by means of restriction fragment analysis to characterize
M. tuberculosis
strains from each country. Chinese, Mongolian, Hong Kong, Filipino, and Korean isolates had comparatively more copies of IS
6110
(proportion with eight or more copies; 95% ± 5%), while Thai, Malaysian, and Vietnamese isolates had fewer copies (proportion with eight or more copies, 60% ± 4%). We found a number of novel IS
1081
types in this study. One IS
1081
type was present in 56% of Filipino isolates, had a specific 6.6-kb
Pvu
II fragment in its IS
6110
DNA fingerprint, and was termed the “Filipino family.” The IS
1081
types of Thai isolates had interposing characteristics between the characteristics of northeastern Asian and southeastern Asian IS
1081
types. A 1.3-kb single-copy IS
6110
fragment was found only in Vietnamese
M. tuberculosis
isolates. Although
M. tuberculosis
isolates from each country had comparatively similar characteristics depending on the classification factor, each country's isolates showed characteristic DNA fingerprints and differed slightly from the isolates from the other countries in either the mode number of IS
6110
copies or the distribution of IS
1081
types.
The 1998 Senegal Epidemic of Meningitis Was Due to the Clonal Expansion of A:4:P1.9, Clone III-1, Sequence Type 5
Neisseria meningitidis
Strains
Authors: Nicolas Pierre , Raphenon Georges , Guibourdenche Martine , Decousset Laurent , Stor Richard , Gaye Abou Beckr
Published: May 2021

Between January and April 1998, a meningitis outbreak due to serogroup A meningococcus took place in Senegal. The outbreak began in Gandiaye, 165 km to the east of Dakar, and progressed towards the towns of Gossas, Niakkhar, Guinguineo, Fatik, Foundiougne, Dioffior, Sokone, Kaolack, and Nioro. At the same time, the outbreak reached regions of Kaffrine, Koungheul, and Tambacounda in the east of Senegal. A total of 1,350 cases and 200 deaths were reported. The WHO Collaborating Center in Marseilles received 24 strains for analysis. All were serogroup A
Neisseria meningitidis
, type 4 and subtype P1.9. Multilocus enzyme electrophoresis, performed by Institut Pasteur Paris, showed that the strains belonged to clone III-1. DNA restriction fragments generated by endonuclease
Bgl
II and analyzed by pulsed-field gel electrophoresis showed 24 indistinguishable fingerprint patterns similar to those of meningococcus strains isolated from African outbreaks since 1988. Three strains were studied by multilocus sequence typing (MLST) with seven loci. The comparison between sequences and existing alleles on the MLST website (
http://mlst.zoo.ox.ac.uk
) allowed us to assign these strains to sequence type 5 (ST5), as their sequences were identical to the consensus at seven loci. All 24 strains were susceptible to penicillin, amoxicillin, chloramphenicol, and rifampin. Subgroup III is finishing its spread towards west of the meningitis belt of Africa. To our knowledge, this is the first time subgroup III, and more precisely ST5, strains are reported as being responsible for a meningitis outbreak in Senegal.
Detection of Clarithromycin-Resistant
Helicobacter pylori
Strains by a Preferential Homoduplex Formation Assay
Authors: Maeda Shin , Yoshida Haruhiko , Matsunaga Hironari , Ogura Keiji , Kawamata Osamu , Shiratori Yasushi , Omata Masao
Published: May 2021

It has been shown that resistance to clarithromycin, a major cause of failure in
Helicobacter pylori
eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with
H. pylori
infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of
H. pylori
infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant
H. pylori
strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.
Comparative Analysis of PCR versus Culture for Diagnosis of Ulceroglandular Tularemia
Authors: Johansson Anders , Berglund Lennart , Eriksson Ulla , Göransson Ingela , Wollin Ralfh , Forsman Mats , Tärnvik Arne , Sjöstedt Anders
Published: May 2021

PCR and culture were comparatively evaluated for their abilities to demonstrate
Francisella tularensis
in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected
F. tularensis
DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an
F. tularensis
-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of
F. tularensis
in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.
Distribution of the Intermedilysin Gene among the Anginosus Group Streptococci and Correlation between Intermedilysin Production and Deep-Seated Infection with
Streptococcus intermedius
Authors: Nagamune Hideaki , Whiley Robert A. , Goto Takatsugu , Inai Yasuko , Maeda Takuya , Hardie Jeremy M. , Kourai Hiroki
Published: May 2021

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by
Streptococcus intermedius
were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in
S. intermedius
and no homologue to the toxin gene is distributed in
S. anginosus
and
S. constellatus
. Thus, the intermedilysin gene is useful as a marker gene of
S. intermedius
. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of
S. intermedius
, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with
S. intermedius
.
Typing of
Candida glabrata
in Clinical Isolates by Comparative Sequence Analysis of the Cytochrome
c
Oxidase Subunit 2 Gene Distinguishes Two Clusters of Strains Associated with Geographical Sequence Polymorphisms
Authors: Sanson Gerdine F. O. , Briones Marcelo R. S.
Published: May 2021

We tested whether comparative sequence analysis of the mitochondrion-encoded cytochrome
c
oxidase subunit 2 gene (
COX2
) could be used to distinguish intraspecific variants of
Candida glabrata
. Mitochondrial genes are suitable for investigation of close phylogenetic relationships because they evolve much faster than nuclear genes, which in general exhibit very limited intraspecific variation. For this survey we used 11 clinical isolates of
C. glabrata
from three different geographical locations in Brazil, 10 isolates from one location in the United States, 1 American Type Culture Collection strain as an internal control, and the published sequence of strain CBS 138. The complete coding region of
COX2
was amplified from total cellular DNA, and both strands were sequenced twice for each strain. These sequences were aligned with published sequences from other fungi, and the numbers of substitutions and phylogenetic relationships were determined. Typing of these strains was done by using 17 substitutions, with 8 being nonsynonymous and 9 being synonymous. Also, cDNAs made from purified mitochondrial polyadenylated RNA were sequenced to confirm that our sequences correspond to the expressed copies and not nuclear pseudogenes and that a frameshift mutation exists in the 3′ end of the coding region (position 673) relative to the
Saccharomyces cerevisiae
sequence and the previously published
C. glabrata
sequence. We estimated the average evolutionary rate of
COX2
to be 11.4% sequence divergence/10
8
years and that phylogenetic relationships of yeasts based on these sequences are consistent with rRNA sequence data. Our analysis of
COX2
sequences enables typing of
C. glabrata
strains based on 13 haplotypes and suggests that positions 51 and 519 indicate a geographical polymorphism that discriminates strains isolated in the United States and strains isolated in Brazil. This provides for the first time a means of typing of
Candida
strains that cause infections by use of direct sequence comparisons and the associated divergence estimates.
Typing of
Histoplasma capsulatum
Isolates Based on Nucleotide Sequence Variation in the Internal Transcribed Spacer Regions of rRNA Genes
Authors: Jiang Bingdong , Bartlett Marilyn S. , Allen Stephen D. , Smith James W. , Wheat L. Joseph , Connolly Patricia A. , Lee Chao-Hung
Published: May 2021

The nucleotide sequences of internal transcribed spacer (ITS) regions of rRNA genes of 24 isolates of
Histoplasma capsulatum
were examined. The results indicate that the sequences of ITS regions in different isolates are not identical. Sequence variations were found at 20 positions in the 496 bp that were sequenced. Ten different sequence patterns, designated types A through H, were observed when the sequences from the 24 isolates were aligned. Twelve isolates from Indianapolis were classified into four different types. Two isolates from New York belonged to type G. Three isolates from different cities were type F. The remaining six isolates were of different types.
Sequence-Based Identification of
Mycobacterium
Species Using the MicroSeq 500 16S rDNA Bacterial Identification System
Authors: Patel Jean Baldus , Leonard Debra G. B. , Pan Xai , Musser James M. , Berman Richard E. , Nachamkin Irving
Published: May 2021

We evaluated the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems), a 500-bp sequence-based identification system, for its ability to identify clinical
Mycobacterium
isolates. The organism identity was determined by comparing the 16S rDNA sequence to the MicroSeq database, which consists primarily of type strain sequences. A total of 113 isolates (18 different species), previously recovered and identified by routine methods from two clinical laboratories, were analyzed by the MicroSeq method. Isolates with discordant results were analyzed by
hsp65
gene sequence analysis and in some cases repeat phenotypic identification, AccuProbe rRNA hybridization (Gen-Probe, Inc., San Diego, Calif.), or high-performance liquid chromatography of mycolic acids. For 93 (82%) isolates, the MicroSeq identity was concordant with the previously reported identity. For 18 (16%) isolates, the original identification was discordant with the MicroSeq identification. Of the 18 discrepant isolates, 7 (six unique sequences) were originally misidentified by phenotypic analysis or the AccuProbe assay but were correctly identified by the MicroSeq assay. Of the 18 discrepant isolates, 11 (seven unique sequences) were unusual species that were difficult to identify by phenotypic methods and, in all but one case, by molecular methods. The remaining two isolates (2%) failed definitive phenotypic identification, but the MicroSeq assay was able to definitively identify one of these isolates. The MicroSeq identification system is an accurate and rapid method for the identification of
Mycobacterium
spp.
VP7 and VP4 Genotyping of Human Group A Rotavirus in Buenos Aires, Argentina
Authors: Argüelles M. H. , Villegas G. A. , Castello A. , Abrami A. , Ghiringhelli P. D. , Semorile L. , Glikmann G.
Published: May 2021

Specific and sensitive tests for the detection and typing of group A rotavirus strains are needed for a more comprehensive knowledge of the epidemiology of rotaviral infection. In this study 500 stool specimens taken from 1996 to 1998 from children with acute diarrhea in Buenos Aires were examined. Group A rotavirus was unequivocally demonstrated in 62% of the samples tested by enzyme-linked immunosorbent assay (ELISA) for detection of VP6 antigen, polyacrylamide gel electrophoresis of double-stranded RNA, and reverse transcription-PCR (RT-PCR) for amplification of the VP7:G (1,062 bp) and VP4:P (876 bp) genes. Only five positive specimens were found by RT-PCR but not by ELISA. G and P typing was carried out by nested amplification of variable sequences of the VP7 and the VP4 genes with six G- and five P-type-specific primers (multiplex PCR). Results obtained by this method showed the prevalence of the following G and P types: G1, 39%; G2, 43%; G4, 4%; P[8], 16%; P[4], 71%. Unexpectedly, the G-P type combination most frequently found was G2P[4] (43%) rather than G1P[8] (12%), which is the most commonly found worldwide. Unusual strains of the type G1P[4] accounted for 14% of the total, while mixed infections with more than one type were found in 10% of the samples. Detection of fecal rotavirus-specific immunoglobulin M (IgM) and IgA antibodies in consecutive samples of two patients taken at daily intervals demonstrated that high levels of IgM and IgA antibodies were detected on day 1 after the onset of disease and that the samples remained positive for about 10 days, after which virus shedding was no longer observed. Multiplex PCR offers a sensitive and specific alternative to determine the prevalence of group A rotavirus G and P types and to identify the emergence of uncommon strains, whereas detection of fecal IgM and IgA antibodies represents a useful supplement to virus detection for the diagnosis of current or recently acquired infections.
Diagnosis of Bubonic Plague by PCR in Madagascar under Field Conditions
Authors: Rahalison L. , Vololonirina E. , Ratsitorahina M. , Chanteau S.
Published: May 2021

The diagnostic value of a PCR assay that amplifies a 501-bp fragment of the
Yersinia pestis caf1
gene has been determined in a reference laboratory with 218 bubo aspirates collected from patients with clinically suspected plague managed in a regional hospital in Madagascar. The culture of
Y. pestis
and the detection of the F1 antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) were used as reference diagnostic methods. The sensitivity of PCR was 89% (57 of 64) for the
Y. pestis
-positive patients, and 80.7% (63 of 78) for the F1 Ag-positive patients. The specificity of PCR for the culture-, F1 Ag-, and antibody-negative patients (
n
= 105) was 100%. Because in Madagascar most patients with plague are managed and their clinical samples are collected in remote villages, the usefulness of PCR was evaluated for routine diagnostic use in the operational conditions of the control program. The sensitivity of PCR was 50% (25 of 50) relative to the results of culture and 35.2% (19 of 54) relative to the results of the F1 Ag immunocapture ELISA. The specificity of PCR under these conditions was 96%. In conclusion, the PCR method was found to be very specific but not as sensitive as culture or the F1 Ag detection method. The limitation in sensitivity may have been due to suboptimal field conditions and the small volumes of samples used for DNA extraction. This technique is not recommended as a routine diagnostic test for plague in Madagascar.
Genotype Profiles of Rotavirus Strains from Children in a Suburban Community in Guinea-Bissau, Western Africa
Authors: Fischer Thea Kølsen , Steinsland Hans , Mølbak Kåre , Ca Rui , Gentsch Jon R. , Valentiner-Branth Palle , Aaby Peter , Sommerfelt Halvor
Published: May 2021

The P (VP4) and G (VP7) genotypes of 167 group A rotavirus strains obtained during the period 1996 to 1998 from 149 children living in a suburban community in Guinea-Bissau, western Africa, were determined by the reverse transcription-PCR technique. A total of nine combinations including five different P types and five different G types were identified. The globally common genotype pairs P[8], G1; P[4], G2; P[8], G3 and P[8], G4 were underrepresented in this study area. We found a substantial year-to-year variation in the occurrence of the genotype combinations. In 1996 and 1997, P[6], G2 was the most frequent, whereas P[8], G1 was more common in 1998. The unusual type P[9], G3 and a few mixed infections were detected. Sixteen percent of the rotavirus-positive samples were nontypeable.
Etiology of Genital Ulcer Disease in Dakar, Senegal, and Comparison of PCR and Serologic Assays for Detection of
Haemophilus ducreyi
Authors: Totten Patricia A. , Kuypers Jane M. , Chen Cheng-Yen , Alfa Michelle J. , Parsons Linda M. , Dutro Susan M. , Morse Stephen A. , Kiviat Nancy B.
Published: May 2021

We used PCR assays to determine the etiology of genital ulcers in patients presenting to a sexually transmitted disease clinic in Dakar, Senegal, and evaluated the ability of two PCR tests (
groEL
and
recD
) and two serological tests (adsorption enzyme immunoassay [EIA] and lipooligosaccharide [LOS] EIA) to detect current
Haemophilus ducreyi
infection. We found that in this population,
H. ducreyi
,
T. pallidum
, and herpes simplex virus HSV DNA were detected in 56, 15, and 13% of 39 genital ulcer specimens, respectively, and
H. ducreyi
DNA was detected in 60% (3 of 5) of samples from ulcerated bubos. Among 40 consecutive patients with genital ulcer disease and with sufficient sample for both PCR assays, the
recD
and
groEL H. ducreyi
PCR assays were 83% concordant, with the
recD
PCR assay detecting six (15%) additional positive specimens and the
groEL
assay detecting one (3%) additional positive specimen. Compared to PCR, the adsorption EIA and LOS EIA tests had sensitivities of 71 and 59% and specificities of 57 and 90%, respectively, for the diagnosis of current
H. ducreyi
infection. While these differences in specificity could be due either to previous infection with
H. ducreyi
or to the detection of cross-reacting antibodies, only 6% of patients from a nearby family planning clinic gave a positive reaction in both the adsorption EIA and LOS EIA assays, indicating that cross-reacting antibodies are not prevalent among clinic attendees in this city. Our studies indicate that the adsorption EIA detects both current and past infection, while the LOS EIA assay is more specific for current infection with
H. ducreyi
in this population.
Prevalence of Toxin Types and Colonization Factors in Enterotoxigenic
Escherichia coli
Isolated during a 2-Year Period from Diarrheal Patients in Bangladesh
Authors: Qadri Firdausi , Das Swadesh Kumar , Faruque A. S. G. , Fuchs George J. , Albert M. John , Sack R. Bradley , Svennerholm Ann-Mari
Published: May 2021

The prevalence of toxin types and colonization factors (CFs) of enterotoxigenic
Escherichia coli
(ETEC) was prospectively studied with fresh samples (
n
= 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (
n
= 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (
P
< 0.001) or LT- and ST-positive (
P
< 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.
Importance of Inoculum Size and Sampling Effect in Rapid Antigen Detection for Diagnosis of
Streptococcus pyogenes
Pharyngitis
Authors: Kurtz Bradley , Kurtz Michael , Roe Martha , Todd James
Published: May 2021

Current recommendations suggest that negative rapid
Streptococcus pyogenes
antigen tests be backed up with a culture, reflecting evidence that culture may have a higher sensitivity and also that testing of a second swab may yield a different (i.e., a positive) result because of variation in sample size or distribution. If the latter is common, the sensitivities of current antigen detection tests might be improved by simply increasing the amount of sample tested. The present study assessed the effect of antigen testing of two swabs extracted together compared to independent testing of each swab extracted separately for children with clinical pharyngitis.
S. pyogenes
grew from one or both swabs for 198 (37%) of 537 children. The combined culture was significantly (
P
< 0.05) more sensitive than culture of either swab alone. Compared to combined culture, antigen testing of two swabs extracted and tested together was significantly more sensitive than two single swab extractions (94.1 versus 80%;
P
= 0.03); however, the specificity was decreased (81.5 versus 89.8 to 92.7%;
P
< 0.05). This study suggests that sample size and/or uneven sample distribution may have influenced the apparent sensitivities of prior studies that compared antigen tests to a single plate culture. A strategy, such as the one used in the present study, that increases the sample size available for antigen testing (i.e., extraction of samples from both swabs) may improve detection rates to a level that will better approximate true disease status and obviate the need for backup cultures if specificity can be improved.
Prevalence of
Campylobacter
,
Arcobacter
,
Helicobacter
, and
Sutterella
spp. in Human Fecal Samples as Estimated by a Reevaluation of Isolation Methods for Campylobacters
Authors: Engberg Jørgen , On Stephen L. W. , Harrington Clare S. , Gerner-Smidt Peter
Published: May 2021

The aims of this study were to investigate the prevalence of campylobacteria including
Campylobacter jejuni
subsp.
jejuni
(
C. jejuni
) and
Campylobacter coli
in human clinical samples and in samples from healthy individuals and to reevaluate the efficacies of conventional selective methods for isolation of
Campylobacter
spp. Two charcoal-based selective media, modified charcoal cefoperazone deoxycholate agar (mCCDA) and cefoperazone-amphotericin-teicoplanin (CAT) agar, were compared with Skirrow's blood-based medium and with a filter method (filter) applied to a yeast-enriched blood agar. A total of 1,376 specimens were tested on all four media, and the percentages of thermophilic
Campylobacter
-positive specimens isolated on Skirrow's medium, filters, CAT agar, and mCCDA were 82, 83, 85, and 95%, respectively. When additional samples were processed with the three selective media, mCCDA recovered significantly more thermophilic
Campylobacter
spp. than Skirrow's medium (
P
= 0.0034). No significant difference between Skirrow's medium and CAT agar was observed in this study. Another six taxa were identified, namely,
Campylobacter concisus
,
Campylobacter curvus
-like bacteria,
Arcobacter butzleri
,
Arcobacter cryaerophilus
,
Helicobacter cinaedi
, and
Sutterella wadsworthensis
. Most of these strains were isolated after 5 to 6 days of incubation by use of the filter technique. This paper provides evidence for the existence of
S. wadsworthensis
in human feces from clinical cases of gastrointestinal disorders and in feces from a healthy individual. Furthermore,
C. concisus
was isolated from a large number of diarrheal cases, particularly those at the extremes of age, but was additionally isolated from the feces of healthy people. Further investigations to establish the role of
C. concisus
and
S. wadsworthensis
in enteric disease is needed. We conclude that a range of campylobacteria may cause infections in Denmark.
Molecular Methods for the Epidemiological Typing of
Salmonella enterica
Serotype Typhi from Hong Kong and Vietnam
Authors: Ling J. M. , Lo N. W. S. , Ho Y. M. , Kam K. M. , Hoa Nguyen Thi Tuyet , Phi Le Thi , Cheng A. F.
Published: May 2021

A total of 217 and 73 strains of
Salmonella enterica
serotype Typhi isolated from 1985 to 1997 in Hong Kong and in 2 months of 1989 and 1990 in Vietnam, respectively, were studied. These isolates were typed by plasmid profile analysis, plasmid fingerprinting, ribotyping with
Pst
I, and total DNA fingerprinting with
Nar
I. There appeared to be no major outbreak of typhoid fever in Hong Kong during the study period since there was considerable heterogeneity among the isolates. Isolates from Hong Kong were different from those from Vietnam. Thirty-seven percent of Vietnamese isolates belonged to two predominant clones, with the rest being heterogeneous in nature. Total DNA fingerprinting supplemented with ribotyping could be a reliable and rapid method for epidemiological typing of
S. enterica
serotype Typhi.
Differentiation of Phylogenetically Related Slowly Growing Mycobacteria by Their
gyrB
Sequences
Authors: Kasai Hiroaki , Ezaki Takayuki , Harayama Shigeaki
Published: May 2021

The conventional methods for identifying mycobacterial species are based on their phenotypic characterization. Since some problematic species are slow growers, their taxonomy takes several weeks or months to identify. The ribosomal DNA (rDNA) sequence-based identification strategy has been adopted to solve this problem. More recently, the
gyrB
sequences have been shown to be useful phylogenetic markers for the identification of species. We determined the
gyrB
sequences of 43 slowly growing strains belonging to 15 species in the genus
Mycobacterium
. The frequencies of base substitutions in the
gyrB
sequences were comparable to those in the 16S-23S rDNA internal transcribed spacer (ITS) sequences. The ITS sequences of four species belonging to the
M. tuberculosis
complex (
M. tuberculosis
,
M. bovis
,
M. africanum
, and
M. microti
) were 100% identical, while four synonymous substitutions were found in the
gyrB
sequences of these strains. Based on the differences found in the
gyrB
sequences, we developed PCR and PCR-restriction fragment length polymorphism methods to discriminate these species.
Evaluation of Whole-Cell and OspC Enzyme-Linked Immunosorbent Assays for Discrimination of Early Lyme Borreliosis from OspA Vaccination
Authors: Wieneke Chad A. , Lovrich Steven D. , Callister Steven M. , Jobe Dean A. , Marks Jennifer A. , Schell Ronald F.
Published: May 2021

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with
Borrelia burgdorferi
. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with
B. burgdorferi
difficult, especially by screening tests based on whole-cell preparations of
B. burgdorferi
. We show that an enzyme-linked immunosorbent assay with
B. burgdorferi
sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with
B. burgdorferi
. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-
B. burgdorferi
sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against
B. burgdorferi
sensu stricto 50772 and OspC, respectively. Although these assays can discriminate
B. burgdorferi
infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.
Prevalence of
Escherichia coli
O157:H7 from Cull Dairy Cows in New York State and Comparison of Culture Methods Used during Preharvest Food Safety Investigations
Authors: McDonough Patrick L. , Rossiter Christine A. , Rebhun Robert B. , Stehman Susan M. , Lein Donald H. , Shin Sang J.
Published: May 2021

A number of protocols for the cultural detection of
Escherichia coli
O157:H7 in clinical fecal specimens have been proposed. In the present study direct plating of cattle feces was compared to three different broth enrichment protocols, i.e., a protocol with modified
E. coli
broth with novobiocin, a protocol with Trypticase soy broth with cefixime and vancomycin, and a protocol with Gram-Negative Broth with novobiocin, for their relative abilities to detect
E. coli
O157:H7 in feces. In all enrichment protocols, dilutions of the enrichment broths onto 150-mm sorbitol-MacConkey agar plates to which cefixime and tellurite were added were used along with reading of agar plates at both 24 and 48 h. Fecal samples came from a preharvest food safety project in which feces from New York cull dairy cattle from a northeastern packing plant along with experimentally inoculated adult dairy cow feces were tested. The performances of the broth enrichments were comparable to each other, but the broth enrichments were superior to direct plating in their ability to detect
E. coli
O157:H7. Regardless of the culture protocol used, recovery of
E. coli
O157:H7 is more likely from fresh fecal specimens than from frozen samples. An overall prevalence of
E. coli
O157:H7 fecal shedding by New York cull dairy cattle of 1.3% was found in specimens just before processing at the packing plant.
Stability of Plasma Human Immunodeficiency Virus Load in VACUTAINER PPT Plasma Preparation Tubes during Overnight Shipment
Authors: Holodniy Mark , Rainen Lynne , Herman Steve , Yen-Lieberman Belinda
Published: May 2021

VACUTAINER PPT plasma preparation tubes were evaluated to determine the effects of various handling and shipping conditions on plasma human immunodeficiency virus (HIV) load determinations. Plasmas obtained from PPT tubes stored and shipped under nine different conditions were compared to conventional EDTA tube plasmas stored at −70°C within 2 h after phlebotomy. Compared to viral loads in frozen EDTA plasma, those in PPT tube plasma that was frozen immediately and either separated or shipped in situ were not significantly different. Viral loads in PPT tube plasma after storage for 6 h at either room temperature or 4°C, followed by shipment at ambient temperature or on wet or dry ice, were not significantly different from baseline viral loads in EDTA or PPT plasma. The results of this study indicate that the HIV load in PPT tube plasma is equivalent to that in standard EDTA plasma. Plasma viral load is not affected by storage or shipment temperature when plasma is collected in PPT tubes. Furthermore, plasmas can be shipped in spun PPT tubes, and the tubes provide a safer and more convenient method for sample collection and transport than regular EDTA tubes.
Comparison ofPasteurellaspp. Simultaneously Isolated from Nasal and Transtracheal Swabs from Cattle with Clinical Signs of Bovine Respiratory Disease
Authors: DeRosa D. C. , Mechor G. D. , Staats J. J. , Chengappa M. M. , Shryock T. R.
Published: May 2021

Twenty-four matched pairs of isolates ofPasteurella haemolyticaand three matched pairs of isolates ofPasteurella multocidawere isolated by using a nasal swab and a transtracheal swab from individual calves with clinical signs of bovine respiratory disease. The identity of each matched pair was confirmed biochemically and serologically. The similarity of the isolates obtained from a nasal swab and from a transtracheal swab was compared by using ribotyping and antibiotic susceptibility analyses. Although the calves were sampled only once with a nasal and a transtracheal swab, when both samples were bacteriologically positive the nasal swab identified the same bacterial species as the transtracheal swab 96% of the time. The nasal swab isolate was genetically identical to the transtracheal isolate in 70% of the matched pairs. Six different ribotypes were observed for theP. haemolyticaisolates, while only one ribotype was observed for the limited number ofP. multocidaisolates. Of the sixP. haemolyticaribotypes, two ribotypes predominated. All the paired isolates displayed similar susceptibility to ceftiofur, erythromycin, tilmicosin, trimethoprim-sulfamethoxazole, and florfenicol, with some minor variations for ampicillin and spectinomycin. These results suggest that a nasal swab culture can be predictive of the bacterial pathogen within the lung when the isolates are from an acutely ill animal and can be used to determine antibiotic susceptibility.
Determination of Antifungal MICs by a Rapid Susceptibility Assay
Authors: Riesselman Marcia H. , Hazen Kevin C. , Cutler Jim E.
Published: May 2021

A novel microtiter assay for antifungal susceptibility testing was developed. This method has several potential advantages over the M27-A assay of the National Committee for Clinical Laboratory Standards. These include provision of MIC results within 6 to 19 h, graphical display of data, and the availability of objective quantitative endpoints. We refer to the method as the rapid susceptibility assay (RSA). RSA is based on substrate utilization by fungi in the presence of antifungal drugs. Substrate uptake is determined by a colorimetric method, which can be scored by analysis of data obtained from a microplate reader. Variables evaluated in the development of the RSA included inoculum size, incubation period, and efficacy with different classes of antifungal drugs and different yeast isolates. With the rapidly available and quantitative endpoints of the RSA, correlation of MICs and therapeutic drug doses can be evaluated more successfully than they can be evaluated by existing assays.
Improved Sensitivity of PCR for Diagnosis of Human Granulocytic Ehrlichiosis Using
epank1
Genes of
Ehrlichia phagocytophila
-Group Ehrlichiae
Authors: Walls Jennifer J. , Caturegli Patrizio , Bakken Johan S. , Asanovich Kristin M. , Dumler J. Stephen
Published: May 2021

The agent of human granulocytic ehrlichiosis (HGE),
Ehrlichia phagocytophila
, and
Ehrlichia equi
probably comprise variants of a single
Ehrlichia
species now called the
Ehrlichia phagocytophila
genogroup. These variants share a unique 153-kDa protein antigen with ankyrin repeat motifs encoded by the
epank1
gene. The
epank1
gene was investigated as an improved target for PCR diagnosis of HGE compared with the currently used 16S rRNA gene target. Primers for
epank1
flanking a region that spans part of the 5′ ankyrin repeat coding region and part of the unique 3′ region were synthesized. Blood samples from 31 patients with suspected HGE who were previously tested by 16S rRNA gene (16S) PCR and indirect immunofluorescent antibody test (IFA) were retrospectively tested with the
epank1
primers. Eleven patients were 16S PCR positive and had a seroconversion detected by IFA (group A), 10 patients were 16S PCR negative but had a seroconversion detected by IFA (group B), and 10 patients were 16S PCR negative and seronegative (group C). Ten of the 11 group A patients were
epank1
PCR positive, all 10 of the group B patients were
epank1
PCR positive, and all of the PCR-negative and seronegative patients (group C) were
epank1
PCR negative. The
epank1
primers are more sensitive than the previously used 16S rRNA gene primers and therefore may be more useful in diagnostic testing for HGE.
Improved Amplification of Genital Human Papillomaviruses
Authors: Gravitt P. E. , Peyton C. L. , Alessi T. Q. , Wheeler C. M. , Coutlée F. , Hildesheim A. , Schiffman M. H. , Scott D. R. , Apple R. J.
Published: May 2021

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83;
P
< 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's χ
2
= 17.2;
P
< 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40.0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.
A Polymorphic Multigene Family Encoding an Immunodominant Protein from
Babesia microti
Authors: Homer M. J. , Bruinsma E. S. , Lodes M. J. , Moro M. H. , Telford S. , Krause P. J. , Reynolds L. D. , Mohamath R. , Benson D. R. , Houghton R. L. , Reed S. G. , Persing D. H.
Published: May 2021

Human babesiosis in the United States is caused predominantly by
Babesia microti
, a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic
B. microti
infection represents a potential threat to the blood supply in areas where
B. microti
is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of
B. microti
(strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of
Plasmodium
. Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific
bmn1-6
homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding
Peromyscus leucopus
(white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of
bmn1-6
does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the
bmn1-6
homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of
B. microti
from the United States and that geographic clustering of subtypes may exist. The nature of the
bmn1-6
gene family suggests a mechanism of antigenic variation in
B. microti
that may occur by recombination, differential expression, or a combination of both mechanisms.
Molecular Cloning and Characterization of the 120-Kilodalton Protein Gene of
Ehrlichia canis
and Application of the Recombinant 120-Kilodalton Protein for Serodiagnosis of Canine Ehrlichiosis
Authors: Yu Xue-jie , McBride Jere W. , Diaz C. Marcela , Walker David H.
Published: May 2021

The 120-kDa outer membrane protein (p120) is a potential adhesin of
Ehrlichia chaffeensis
, and recombinant p120 is very useful for serodiagnosis of human monocytotropic ehrlichiosis. The analogous gene of p120 in
Ehrlichia canis
was cloned, sequenced, and expressed. Like the
E. chaffeensis
p120, the
E. canis
p120 contains tandem repeat units. However, neither the repeat number nor the amino acid sequences in the repeats are identical in the two
Ehrlichia
species. The repeat units are hydrophilic and by probability analysis are predicted to be surface exposed in both species. The repeat regions of the p120s of the two species have common amino acid sequences that are predicted to be surface exposed. The overall amino acid sequence of the
E. canis
p120 is 30% homologous to that of
E. chaffeensis
p120. Protein immunoblotting demonstrated that the recombinant
E. canis
p120 reacted with convalescent sera from dogs with canine ehrlichiosis. These results indicate that the recombinant p120 is a potential antigen for the serodiagnosis of canine ehrlichiosis.