bluuebunny/crossref_metadata_2025 huggingface dataset:
The
Yersinia enterocolitica
Motility Master Regulatory Operon,
flhDC
, Is Required for Flagellin Production, Swimming Motility, and Swarming Motility
Authors: Young Glenn M. , Smith Michael J. , Minnich Scott A. , Miller Virginia L.
Published: December 2019

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen
Yersinia enterocolitica
can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as
flgEF
,
flgMN
,
flhBA
, and
fliA
, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by
Y. enterocolitica
to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon,
flhDC
, was cloned. Mutations within
flhDC
completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of
flhDC
indicated that it is required for the production of flagella. When
flhDC
was expressed from an inducible p
tac
promoter, flagellin production was shown to be dependent on levels of
flhDC
expression. Phenotypically, induction of the p
tac-flhDC
fusion also corresponded to increased levels of both swimming and swarming motility.
DNA Polymerase II (
polB
) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links in
Escherichia coli
Authors: Berardini Mark , Foster Patricia L. , Loechler Edward L.
Published: December 2019

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into
Escherichia coli
, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a
polB
+
(∼0.6) than in a Δ
polB
(∼0.1) strain. Complementation with a
polB
+
-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments,
E. coli
was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <
polB
<
recA
<
polB recA
∼
uvrA
. Because cells deficient in either recombination (
recA
) or DNA polymerase II (
polB
) are hypersensitive to nitrogen mustard killing,
E. coli
appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in
E. coli
. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.
In Vitro Transcriptional Studies of the
bkd
Operon of
Pseudomonas putida
:
l
-Branched-Chain Amino Acids and
d
-Leucine Are the Inducers
Authors: Madhusudhan Kunapuli T. , Luo Jinhe , Sokatch John R.
Published: December 2019

BkdR is the transcriptional activator of the
bkd
operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of
Pseudomonas putida
. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5′ region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the
bkd
operon was obtained by using a vector containing the
bkdR-bkdA1
intergenic region plus the putative ρ-independent terminator of the
bkd
operon. Substrate DNA, BkdR, and any of the
l
-branched-chain amino acids or
d
-leucine was required for transcription. Branched-chain keto acids,
d
-valine, and
d
-isoleucine did not promote transcription. Therefore, the
l
-branched-chain amino acids and
d
-leucine are the inducers of the
bkd
operon. The concentration of
l
-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the
bkd
operon by BkdR during enzyme induction which incorporates these results is presented.
Ribosomal −1 Frameshifting during Decoding ofBacillus subtilis cddOccurs at the Sequence CGA AAG
Authors: Mejlhede Nina , Atkins John F. , Neuhard Jan
Published: December 2019

During translation of theBacillus subtilis cddgene, encoding cytidine deaminase (CDA), a ribosomal −1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when thecddgene is expressed from a multicopy plasmid inEscherichia coliand when it is expressed from the chromosomal copy inB. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. Thecddgene was cloned in pUC19 such that thelacZ′ gene of the vector followed thecddgene in the −1 reading frame immediately after thecddstop codon. By using site-directed mutagenesis of thecdd-lacZ′ fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-framecddstop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed.
Synthesis of the ς
D
Protein Is Not Sufficient To Trigger Expression of Motility Functions in
Bacillus subtilis
Authors: Yang Dong-Hui , von Kalckreuth Johannes , Allmansberger Rudolf
Published: December 2019

The gene encoding ς
D
,
sigD
, is transcribed from two promoter regions, the
fla/che
promoter region in front of the
fla/che
operon and P

sigD

directly in front of
sigD
. If ς
D
is translated from transcripts originating from P

sigD

, the cell is unable to express motility functions but synthesizes autolysins. Therefore, one function of the additional promoter is to allow the cell to express autolysins without expressing motility functions as well.
Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis
Authors: Levett Paul N. , Whittington Carol U.
Published: December 2019

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.
Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences
Authors: Roth Andreas , Fischer Marga , Hamid Mohamed E. , Michalke Sabine , Ludwig Wolfgang , Mauch Harald
Published: December 2019

Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in
Mycobacterium kansasii
and
M. gastri
, or highly similar, for example, in
M. malmoense
and
M. szulgai
. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus
Mycobacterium
representing 13 species (
M. avium
,
M. conspicuum
,
M. gastri
,
M. genavense
,
M. kansasii
,
M. malmoense
,
M. marinum
,
M. shimoidei
,
M. simiae
,
M. szulgai
,
M. triplex
,
M. ulcerans
, and
M. xenopi
). An alignment of these sequences together with additional sequences available in the EMBL database (for
M. intracellulare
,
M. phlei
,
M. smegmatis
, and
M. tuberculosis
) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for
M. simiae
,
M. genavense
, and
M. triplex
, and distinct branches for
M. xenopi
and
M. shimoidei
. Comparisons of
M. gastri
with
M. kansasii
and
M. malmoense
with
M. szulgai
revealed ITS sequence similarities of 93 and 88%, respectively.
M. marinum
and
M. ulcerans
possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.
Use of Ribotyping and Hemolysin Activity To Identify Highly Virulent
Streptococcus suis
Type 2 Isolates
Authors: Staats Jacque J. , Plattner Brandon L. , Nietfeld Jerome , Dritz Steve , Chengappa M. M.
Published: December 2019

Nineteen
Streptococcus suis
type 2 isolates were evaluated for their virulence in pigs and mice. Of these, seven were determined to be highly virulent in pigs on the basis of clinical sign scores and gross pathology and histopathology results. Clinical sign scores correlated with gross pathology and histopathology scores at
P
equal to 0.004 and
P
equal to 0.009, respectively. The virulence of highly virulent isolates in pigs compared somewhat with virulence in mice, but the correlation was not significant. No correlation of virulence was noted among the moderately virulent and avirulent isolates in pigs and mice. Chromosomal DNAs from all
S. suis
isolates were evaluated by
Pst
I,
Pvu
II,
Eco
RI, and
Hae
III restriction enzyme digestion followed by hybridization with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from 16S and 23S rRNAs from
Escherichia coli
. The hybridization patterns (ribotypes) varied depending upon the enzyme used, but a significant number of isolates determined to be highly virulent in pigs had unique hybridization patterns compared with those of the moderately virulent and avirulent isolates (
P
= 0.002). In addition, hemolysin activity showed a high correlation to virulence (
P
= 0.00008) and ribotype (
P
= 0.002).
Comparative Analysis of Infrequent-Restriction-Site PCR and Pulsed-Field Gel Electrophoresis for Epidemiological Typing of
Legionella pneumophila
Serogroup 1 Strains
Authors: Riffard Serge , Presti François Lo , Vandenesch François , Forey Françoise , Reyrolle Monique , Etienne Jerome
Published: December 2019

Two methods were compared for the analysis of 48 unrelated and epidemiologically related
Legionella pneumophila
serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for
Xba
I and
Pst
I restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with
Sfi
I. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of
L. pneumophila
from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, ≥0.996) tool for typing of
Legionella
. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.
Transmission of Ovine Herpesvirus 2 in Lambs
Authors: Li Hong , Snowder Garry , O’Toole Donal , Crawford Timothy B.
Published: December 2019

The pattern of acquisition of ovine herpesvirus 2 (OHV-2) infection in lambs was examined by a competitive-inhibition enzyme-linked immunosorbent assay and PCR. Newborn lambs (
n
= 118) did not exhibit antibody at birth. Viral DNA in peripheral blood leukocytes was detected in only 3% (
n
= 77) of newborn lambs before suckling. After nursing, viral DNA was sporadically present in about 10 to 20% of lambs until about 3 months of age. Thereafter, strong DNA signals began to appear in increasing numbers of lambs, reaching 100% by 5.5 months of age. Viral DNA in nasal secretions began to be detectable in about 30% of lambs at 5.5 months of age, achieved significant levels in 88% of lambs by 7.5 months of age, and then declined. The kinetics of the humoral immune response in lambs paralleled those of viral DNA in nasal secretions but did not parallel its presence in blood leukocytes. In the experiment to define the time of infection of OHV-2 in lambs, all five lambs separated from the flock at 2.5 months of age remained uninfected until the termination of the experiment at 1 year of age. In contrast, lambs weaned at 2.5 months of age and returned to the flock had become infected at 3.5 months of age. Weaning and separation from the flock at 3.5 months of age did not prevent infection. The study showed that OHV-2 infection does not commonly occur in perinatal lambs and that OHV-2-free sheep can be established by separation of lambs at the proper time, which has important implications for potential control measures.
Detection and Confirmation of
Mycoplasma pneumoniae
in Urogenital Specimens by PCR
Authors: Sharma S. , Brousseau R. , Kasatiya S.
Published: December 2019

Following the isolation of
Mycoplasma pneumoniae
from urogenital specimens (M. Goulet, R. Dular, J. G. Tully, G. Billows, and S. Kasatiya, J. Clin. Microbiol. 33:2823–2825, 1995), a study was undertaken to confirm the observations by PCR. Specific primers directed to the P1 adhesin gene of
M. pneumoniae
were used. A total of 300 genital specimens were tested for
M. pneumoniae
and
Mycoplasma genitalium
by culture and PCR. Of these, 15 were positive by culture and 17 were positive by PCR for
M. pneumoniae
. No
M. genitalium
was detected in any of the specimens by either method. The present study demonstrates that PCR is sensitive and rapid compared to cumbersome culture methods and can be used to detect
M. pneumoniae
in urogenital specimens in a routine diagnostic laboratory.
Improving the Yield of Blood Cultures for Patients with Early Lyme Disease
Authors: Wormser Gary P. , Nowakowski John , Nadelman Robert B. , Bittker Susan , Cooper Denise , Pavia Charles
Published: December 2019

This study was designed to improve the recovery of
Borrelia burgdorferi
from blood. With the techniques used,
B. burgdorferi
could be recovered from the blood of approximately 25% of patients with early Lyme disease associated with erythema migrans. Serum was a better source of culture material than whole blood. The volume of blood cultured correlated directly with yield, particularly for patients with a single erythema migrans lesion.
Results from 5 Years of Nationwide DNA Fingerprinting of
Mycobacterium tuberculosis
Complex Isolates in a Country with a Low Incidence of
M. tuberculosis
Infection
Authors: Bauer Jeanett , Yang Zhenhua , Poulsen Sigrid , Andersen Åse B.
Published: December 2019

Results from DNA fingerprint analyses of
Mycobacterium tuberculosis
complex isolates from tuberculosis (TB) patients diagnosed during 5 years in Denmark are presented. The lack of success in eradicating TB in this low-incidence country may be explained by an unrecognized high frequency of active TB transmission (57%) among native Danes. Only two strains of
M. tuberculosis
are responsible for 40% of all clustered cases of TB among Danes.
Multicomponent Chimeric Antigen for Serodiagnosis of Canine Visceral Leishmaniasis
Authors: Soto Manuel , Requena Jose M. , Quijada Luis , Alonso Carlos
Published: December 2019

In this work, we describe the assembly of a synthetic gene coding for several antigenic determinants found in different
Leishmania infantum
antigens. Selected epitopes were derived from the ribosomal proteins LiP2a, LiP2b, and LiP0 and from the histone H2A. The resulting gene was overexpressed in
Escherichia coli
either as a fusion protein (with the vector pMAL-c2) or alone (with the vector pQE). In both cases, high-level bacterial production of the recombinant protein was achieved and the products were found to be stable. Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments confirmed that the corresponding epitopes are present in the engineered protein. Finally, a serological evaluation of this multiple-epitope protein by Falcon assay screening test-ELISA revealed a sensitivity of 79 to 93% and a specificity of 96 to 100% in diagnosis of canine visceral leishmaniasis, indicating that this protein represents a valuable tool for serodiagnosis.
Ultrasensitive Reverse Transcription-PCR Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Sun Rita , Ku Joanne , Jayakar Harsha , Kuo Jo-Chi , Brambilla Donald , Herman Steven , Rosenstraus Maurice , Spadoro Joanne
Published: December 2019

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This “ultrasensitive” method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.
Genetic Analysis of
Histoplasma capsulatum
Strains Isolated from Clinical Specimens in Thailand by a PCR-Based Random Amplified Polymorphic DNA Method
Authors: Poonwan Natteewan , Imai Tamae , Mekha Nanthawan , Yazawa Katsukiyo , Mikami Yuzuru , Ando Akikazu , Nagata Yoshiho
Published: December 2019

Thirteen strains of
Histoplasma capsulatum
were isolated from clinical specimens, including those from AIDS patients, in Thailand. Random amplified polymorphic DNA (RAPD) analysis with three different PCR primers showed that the DNA fingerprint patterns of the Thai isolates were very similar to each other and homogeneous, with only one exceptional strain, although the patterns were clearly different from those of a reference North American strain with all primers tested. Although the difference in the DNA fingerprinting patterns was minor, Thai isolates could be classified into two to four groups. A common PCR band (about 700 bp) in the patterns of all
H. capsulatum
strains was extracted, and its DNA sequence was determined. A new PCR primer set for the identification of
H. capsulatum
species was developed based on this sequence information. This primer set was 100% successful in the identification of the reference strain as well as all Thai isolates. The results of specificity tests of the primer set for the identification of the fungus are also discussed.
Molecular Epidemiology of an Outbreak of
Enterobacter cloacae
in the Neonatal Intensive Care Unit of a Provincial Hospital in Gauteng, South Africa
Authors: van Nierop W. H. , Duse A. G. , Stewart R. G. , Bilgeri Y. R. , Koornhof H. J.
Published: December 2019

An outbreak of
Enterobacter cloacae
in the neonatal intensive care unit of a provincial hospital in Gauteng, South Africa, resulting in nine deaths was investigated. Macrorestriction analysis using pulsed-field gel electrophoresis revealed that three isolates of
E. cloacae
from blood cultures of patients, six from environmental sources, and one from the hands of a staff member belonged to the same genotypic cluster.
Description of
Gemella sanguinis
sp. nov., Isolated from Human Clinical Specimens
Authors: Collins Matthew D. , Hutson Roger A. , Falsen Enevold , Sjöden Berit , Facklam Richard R.
Published: December 2019

Six strains of a hitherto undescribed gram-positive, catalase-negative, facultatively anaerobic coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains were genealogically identical and constitute a new subline within the genus
Gemella
. The unknown bacterium was readily distinguished from
Gemella haemolysans
,
Gemella bergeriae
, and
Gemella morbillorum
by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as
Gemella sanguinis
sp. nov. The type strain is CCUG 37820
T
.
Diagnosis of
Mycoplasma pneumoniae
Pneumonia in Children
Authors: Waris Matti E. , Toikka Pia , Saarinen Taina , Nikkari Simo , Meurman Olli , Vainionpää Raija , Mertsola Jussi , Ruuskanen Olli
Published: December 2019

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of
Mycoplasma pneumoniae
infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for
M. pneumoniae
-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of
M. pneumoniae
DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of
M. pneumoniae
pneumonia in children of any age.
Prevalence of Human Calicivirus Infections in Kenya as Determined by Enzyme Immunoassays for Three Genogroups of the Virus
Authors: Nakata Shuji , Honma Shinjiro , Numata Kazuko , Kogawa Keiko , Ukae Susumu , Adachi Noriaki , Jiang Xi , Estes Mary K. , Gatheru Zippora , Tukei Peter M. , Chiba Shunzo
Published: December 2019

An epidemiological survey on human calicivirus (HuCV) infections and associated gastroenteritis in infants was conducted to clarify the prevalence of HuCV infections in infants and adults in Kenya. Enzyme immunoassays (EIAs) for three genogroups of HuCVs, Norwalk virus (NV), Mexico virus (MXV), and Sapporo virus (SV), were used to detect antigen or antibody. We tested 1,431 stool samples obtained from children younger than 6 years old with acute gastroenteritis who visited outpatient clinics in three districts in Kenya from August 1991 to July 1994. Thirty-two (2.2%) of these stool samples were positive for SV antigen. Only one (0.1%) of 1,186 samples was positive for NV antigen and none of 246 samples was positive for MXV antigen. One hundred ninety-three serum samples were tested for antibodies to NV and MXV, and 64 of them were examined for antibody to SV. The pattern of the age-related prevalence of serum antibody to NV was different from that of antibodies to MXV and SV. The acquisition of serum antibodies to HuCVs in the three genogroups appeared in early childhood, at about 1 to 2 years of age. The prevalence of serum antibody to NV was low (about 60%) throughout adulthood compared with a high prevalence of antibody (∼80 to 90%) to MXV and SV. These data indicate that infections with viruses in the three genogroups of HuCVs are common in Kenya, and immunological responses to NV may be different from those to MXV and SV. The EIAs for the detection of NV and MXV antigens appear to be quite specific for prototype NV and MXV strains, respectively, so that they can detect only a few strains of HuCVs related to them. Alternatively, NV and MXV caused less severe infections that did not bring children to the outpatient clinics for gastroenteritis in Kenya.
Nucleic Acid Sequence-Based Amplification, a New Method for Analysis of Spliced and Unspliced Epstein-Barr Virus Latent Transcripts, and Its Comparison with Reverse Transcriptase PCR
Authors: Brink Antoinette A. T. P. , Vervoort Marcel B. H. J. , Middeldorp Jaap M. , Meijer Chris J. L. M. , van den Brule Adriaan J. C.
Published: December 2019

Nucleic acid sequence-based amplification (NASBA) assays were developed for direct detection of Epstein-Barr virus (EBV) transcripts encoding EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and
Bam
HIA rightward frame 1 (BARF1) and for the noncoding EBV early RNA 1 (EBER1). The sensitivities of all NASBAs were at least 100 copies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA assays. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clinical material. BARF1 expression was found in 6 of 7 nasopharyngeal carcinomas (NPC) but in 0 of 22 Hodgkin’s disease (HD) cases, whereas LMP2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For detection of EBNA1 transcripts in HLs (
n
= 12) and T- and B-cell non-Hodgkin’s lymphomas (
n
= 3 and
n
= 2, respectively), NASBA was compared with reverse transcriptase (RT) PCR. Two samples were positive only with NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We conclude that NASBA is suitable for sensitive and specific detection of the above-mentioned EBV transcripts, regardless of their splicing patterns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs developed in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.
Diagnostic Potential of Toscana Virus N Protein Expressed in
Escherichia coli
Authors: Valassina Marcello , Soldateschi Dario , Dal Maso Grazia Maria , Santini Laura , Bianchi Silvia , Valensin Pier Egisto , Cusi Maria Grazia
Published: December 2019

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in
Escherichia coli
by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (
r
= 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.
Detection and Characterization of Novel Rotavirus Strains in the United States
Authors: Ramachandran M. , Gentsch J. R. , Parashar U. D. , Jin S. , Woods P. A. , Holmes J. L. , Kirkwood C. D. , Bishop R. F. , Greenberg H. B. , Urasawa S. , Gerna G. , Coulson B. S. , Taniguchi K. , Bresee J. S. , Glass R. I. ,   the National Rotavirus Strain Surveillance System Collaborating Laboratories
Published: December 2019

We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0.3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.
Rapid Identification of
Candida
Species with Species-Specific DNA Probes
Authors: Elie Cheryl M. , Lott Timothy J. , Reiss Errol , Morrison Christine J.
Published: December 2019

Rapid identification of
Candida
species has become more important because of an increase in infections caused by species other than
Candida albicans
, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common
Candida
species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional
Candida
species (
C. guilliermondii
,
C. kefyr
,
C. lambica
,
C. lusitaniae
,
C. pelliculosa
,
C. rugosa
, and
C. zeylanoides
) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (
C. haemulonii
,
C. norvegica
,
C. norvegensis
,
C. utilis
, and
C. viswanathii
), and a probe for the newly described species
C. dubliniensis
, creating a panel of 18
Candida
species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as
C. albicans
by the currently available API 20C database but determined to be
C. dubliniensis
by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying
Candida
species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species,
C. dubliniensis
, from
C. albicans
.
Use of the BacT/Alert Blood Culture System for Culture of Sterile Body Fluids Other than Blood
Authors: Bourbeau Paul , Riley Julie , Heiter Barbara J. , Master Ron , Young Carol , Pierson Carl
Published: December 2019

Studies have demonstrated that large-volume culture methods for sterile body fluids other than blood increase recovery compared to traditional plated-medium methods. BacT/Alert is a fully automated blood culture system for detecting bacteremia and fungemia. In this study, we compared culture in BacT/Alert standard aerobic and anaerobic bottles, BacT/Alert FAN aerobic and FAN anaerobic bottles, and culture on routine media for six specimen types, i.e., continuous ambulatory peritoneal dialysate (CAPD), peritoneal, amniotic, pericardial, synovial, and pleural fluids. Specimen volumes were divided equally among the three arms of the study. A total of 1,157 specimens were tested, with 227 significant isolates recovered from 193 specimens. Recovery by method was as follows: standard bottles, 186 of 227 (82%); FAN bottles, 217 of 227 (96%); and routine culture, 184 of 227 (81%). The FAN bottles recovered significantly more gram-positive cocci (
P
< 0.001),
Staphylococcus aureus
(
P
= 0.003), coagulase-negative staphylococci (
P
= 0.008), gram-negative bacilli (
P
< 0.001),
Enterobacteriaceae
(
P
= 0.005), and total organisms (
P
< 0.001) than the routine culture. There were no significant differences in recovery between the standard bottles and the routine culture. The FAN aerobic bottle recovered significantly more gram-positive cocci (
P
< 0.001),
S. aureus
isolates (
P
< 0.001), coagulase-negative staphyococci (
P
= 0.003), and total organisms (
P
< 0.001) than the standard aerobic bottle, while the FAN anaerobic bottle recovered significantly more gram-positive cocci (
P
< 0.001),
S. aureus
isolates (
P
< 0.001),
Enterobacteriaceae
(
P
= 0.03), and total organisms (
P
< 0.001) than the standard anaerobic bottle. For specific specimen types, significantly more isolates were recovered from the FAN bottles compared to the routine culture for synovial (
P
< 0.001) and CAPD (
P
= 0.004) fluids. Overall, the FAN bottles were superior in performance to both the standard bottles and the routine culture for detection of microorganisms from the types of sterile body fluids included in this study.
Endocarditis and Aortal Embolization Caused by
Aspergillus terreus
in a Patient with Acute Lymphoblastic Leukemia in Remission: Diagnosis by Peripheral-Blood Culture
Authors: Schett G. , Casati B. , Willinger B. , Weinländer G. , Binder T. , Grabenwöger F. , Sperr W. , Geissler K. , Jäger U.
Published: December 2019

Disseminated infection with
Aspergillus terreus
is a rare disease that affects only the immunocompromised host. We report a case of systemic infection with
A. terreus
resulting in endocarditis, aortic embolization, and splenic infarction in a patient with acute lymphoblastic leukemia. Diagnosis through peripheral blood culture, lack of pulmonary involvement, and onset of disease during complete remission from leukemia constitute uncommon features of this case.
Distribution of Hepatitis B Virus Genotypes in Two Different Pediatric Populations from Argentina
Authors: Mbayed V. A. , López J. L. , Telenta P. F. S. , Palacios G. , Badía I. , Ferro A. , Galoppo C. , Campos R.
Published: December 2019

Differences in pathogenesis and the probability of becoming a chronic carrier depend on the age at which hepatitis B virus (HBV) infection is acquired, ranging from 82% in infants less than 6 months of age to 15 to 30% in older children. HBV genotypes from 22 pediatric patients from two areas that differ in prevalence have been determined. Phylogenetic analysis shows a clear difference between the genotype distribution in Buenos Aires, a low-prevalence area, and that found in Gualeguay, Entre Rı́os, a high-prevalence area. While the analysis allocated the sequences in the Buenos Aires group to genotypes A (36%), D (9%), and F (55%), the Gualeguay group presented exclusively genotype A isolates with very low nucleotide divergence, which suggests a strong founder viral population. The high prevalence of genotype F in the Buenos Aires group and its high intragroup heterogeneity agree with the American origin of this genotype.
Rapid and Sensitive Assay for Detection of Enterotoxigenic
Bacteroides fragilis
Authors: Zhang Guangming , Weintraub Andrej
Published: December 2019

Bacteroides fragilis
is an obligatory anaerobic, gram-negative bacterium found among the normal intestinal flora of humans. Enterotoxigenic strains of
B. fragilis
(ETBF) have been associated with diarrheal diseases in humans and animals. The enterotoxin of ETBF induces fluid changes in ligated intestinal segments and cytotoxic response in HT29/C1 cells. By using a pair of monoclonal antibodies (MAbs; MAb C3 and MAb 4H8) specific for the lipopolysaccharide of
B. fragilis
, an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) was developed. After DNA extraction, a 294-bp fragment was amplified. The specificity of the IMS-PCR assay was evaluated by adding previously isolated and confirmed ETBF strains to normal fecal samples. All fecal samples to which ETBF strains were added were positive, showing a 100% specificity. The spiked fecal samples were also used for evaluation of the sensitivity of the assay. The detection limit was found to be ∼50 CFU/g of feces. By this method 10 clinical fecal samples (5 from patients with diarrhea and 5 from healthy controls) were examined. The results of PCR were in accordance with the results of the HT29/C1 cell assay for all samples. The minimum time to retrieval of the final result by the IMS-PCR method is 36 h. The proposed IMS-PCR assay is rapid and sensitive for the direct detection of ETBF in stool samples.
Differentiation of
Campylobacter jejuni
Serotype O19 Strains from Non-O19 Strains by PCR
Authors: Misawa Naoaki , Allos Ban Mishu , Blaser Martin J.
Published: December 2019

Guillain-Barré syndrome (GBS), a neurologic disease characterized by acute paralysis, is frequently preceded by
Campylobacter jejuni
infection. Serotype O19 strains are overrepresented among GBS-associated
C. jejuni
isolates. We previously showed that all O19 strains tested were closely related to one another by randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism analyses. RAPD analysis demonstrated a 1.4-kb band in all O19 strains tested but in no non-O19 strains. We cloned this O19-specific band; nucleotide sequence analysis revealed a truncated open reading frame with significant homology to DNA gyrase subunit B (
gyrB
) of
Helicobacter pylori
. PCR using the random primer and a primer specific for
gyrB
showed that in non-O19 strains, the random primer did not recognize the downstream
gyrB
binding site. The regions flanking each of the random primer binding sites were amplified by degenerate PCR for further sequencing. Although the random primer had several mismatches with the downstream
gyrB
binding site, a single nucleotide polymorphism 6 bp upstream from the 3′ terminus was found to distinguish O19 and non-O19 strains. PCR using 3′-mismatched primers based on this polymorphism was designed to differentiate O19 strains from non-O19 strains. When a total of 42 (18 O19 and 24 non-O19) strains from five different countries were examined, O19 strains were distinguishable from non-O19 strains in each case. This PCR method should permit identification of O19
C. jejuni
strains.
Armored RNA Technology for Production of Ribonuclease-Resistant Viral RNA Controls and Standards
Authors: Pasloske Brittan L. , Walkerpeach Cindy R. , Obermoeller R. Dawn , Winkler Matthew , DuBois Dwight B.
Published: December 2019

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. “Armored RNA” is a complex of MS2 bacteriophage coat protein and RNA produced in
Escherichia coli
by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1)
gag
gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at −20°C or for 60 days at 4°C. Additionally, Armored RNA preparations are homogeneous and noninfectious.
Comparison of Two Measures of Human Immunodeficiency Virus (HIV) Type 1 Load in HIV Risk Groups
Authors: Lyles Cynthia M. , Vlahov David , Farzadegan Homayoon , Astemborski Jacquie , Margolick Joseph B. , Masters Beth A. , Schroeder Jennifer , Quinn Thomas C.
Published: December 2019

Levels of viral burden were compared across risk group and gender populations among 485 human immunodeficiency virus type 1 (HIV-1)-infected participants consisting of 190 male injection drug users (IDUs), 92 female IDUs, and 203 homosexual men. Viral burden was quantified by a microculture technique to determine cell-associated infectious units per 10
6
peripheral blood mononuclear cells (IUPM) and by reverse transcriptase PCR (Amplicor) to determine plasma HIV RNA levels. Adjusting for CD4
+
cell count, females had a lower infectious HIV load than all males combined (0.33 log
10
lower;
P
= 0.004), and homosexual men had a 0.29 log
10
higher infectious viral load than all IDUs combined (
P
= 0.001). For HIV RNA levels, females had lower levels than males (0.19 log
10
lower;
P
= 0.04), but no differences were observed by risk group. After controlling for percent CD4
+
cells, no differences were found by risk group for either assay, but females still had a 0.25 log
10
lower infectious viral load than males (
P
= 0.04) and a viral RNA load similar to that of males (
P
= 0.25). The correlation between infectious viral load and HIV RNA load was 0.58 overall, which did not differ by gender or risk group. Our data suggest that differences in viral load may exist by gender and that any differences observed by risk group are driven predominantly by gender or percent CD4
+
cell differences. These data also confirm a moderate correlation between cell-associated infectious viral load and plasma HIV RNA load, which appears to be similar by gender and across risk groups.
Incidental Finding of
Myxobolus
Spores (Protozoa: Myxozoa) in Stool Samples from Patients with Gastrointestinal Symptoms
Authors: Boreham R. E. , Hendrick S. , O’Donoghue P. J. , Stenzel D. J.
Published: December 2019

Myxozoan spores were detected in fecal samples from three patients presenting with abdominal pain and/or diarrhea. The spores were identical to those of
Myxobolus plectroplites
, a previously described pathogen from the freshwater fish
Plectroplites ambiguus
. All patients had recently eaten fish caught from local waters, and frozen fillets of such fish were found to be infected with
M. plectroplites
cysts. The passage of spores unchanged through the alimentary tract suggests they were incidental findings unrelated to clinical symptoms, especially since other enteric pathogens were present in two patients.
Detection of Specific Antibodies in Saliva during Dengue Infection
Authors: Cuzzubbo Andrea J. , Vaughn David W. , Nisalak Ananda , Suntayakorn Saroj , Aaskov John , Devine Peter L.
Published: December 2019

Saliva was collected prospectively from patients presenting with suspected dengue infection 4 to 8 days after the onset of symptoms and assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA). Laboratory diagnosis was based on virus isolation and on hemagglutination inhibition (HAI) assay and an in-house IgM and IgG capture ELISA. With a positive result defined as either salivary IgM or IgG levels above the cutoff value, an overall sensitivity of 92% was obtained for both primary- and secondary-dengue patients (22 of 24), while no patients with non-flavivirus infections (
n
= 11) and no healthy laboratory donors (
n
= 17) showed elevation of salivary antidengue antibody (100% specificity). Salivary IgG levels correlated well with serum HAI titer (
r
= 0.78), and salivary IgG levels could be used to distinguish between primary- and secondary-dengue virus infections.