bluuebunny/crossref_metadata_2025 huggingface dataset:
Multicenter Comparison of Roche COBAS AMPLICOR MONITOR Version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex Version 3.0 for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Murphy Donald G. , Côté Louise , Fauvel Micheline , René Pierre , Vincelette Jean
Published: December 2019

The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1.5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (
P
< 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log
10
RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log
10
lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.
Real-Time PCR for Quantitative Detection of
Toxoplasma gondii
Authors: Lin Mei-Hui , Chen Tse-Ching , Kuo Tseng-tong , Tseng Ching-Chung , Tseng Ching-Ping
Published: December 2019

The protozoan
Toxoplasma gondii
is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of
T. gondii
. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the
T. gondii
B1 gene. After 40 PCR cycles, the cycle threshold values (C
T
) indicative of the quantity of the target gene were determined. Typically, a C
T
of 25.09 was obtained with DNA from 500 tachyzoites of the
T. gondii
RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05
T. gondii
tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C
T
of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of
T. gondii
. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.
Genetic Diversity and Clonal Patterns among Antibiotic-Susceptible and -Resistant
Streptococcus pneumoniae
Colonizing Children: Day Care Centers as Autonomous Epidemiological Units
Authors: Sá-Leão Raquel , Tomasz Alexander , Sanches Ilda Santos , Nunes Sónia , Alves C. Rute , Avô António Brito , Saldanha Joana , Kristinsson Karl G. , de Lencastre Hermínia
Published: December 2019

Characterization by antibiotype of the 1,096
Streptococcus pneumoniae
recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread
S. pneumoniae
clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.
Characterization of aBorrelia burgdorferiVlsE Invariable Region Useful in Canine Lyme Disease Serodiagnosis by Enzyme-Linked Immunosorbent Assay
Authors: Liang Fang Ting , Jacobson Richard H. , Straubinger Reinhard K. , Grooters Amy , Philipp Mario T.
Published: December 2019

Sera collected from dogs experimentally infected withBorrelia burgdorferiby tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR1to IR6) of VlsE, the variable surface antigen ofB. burgdorferi. Six synthetic peptides (C1to C6), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR2and IR6, were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR6appears earlier and is stronger than that to IR2. Thus, the IR6sequence alone appeared to be sufficient for serodiagnosis. When C6alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n= 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C6ELISA, compared to 20 positives detected by immunoblot analysis using culturedB. burgdorferilysates as antigen. The sensitivity of using C2and C6together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C6alone, confirming that C6suffices as a diagnostic probe. Moreover, the C6ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other thanB. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C6ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.
Marked Differences in Quantity of Infectious Human Immunodeficiency Virus Type 1 Detected in Persons with Controlled Plasma Viremia by a Simple Enhanced Culture Method
Authors: Tremblay Cécile L. , Giguel Françoise , Merrill Debra P. , Wong Johnson T. , Rosenberg Eric , Kalams Spyros , Walker Bruce D. , D'Aquila Richard T. , Hirsch Martin S.
Published: December 2019

Culture of autologous CD4 lymphocytes from peripheral blood mononuclear cells compared favorably with two other methods for the measurement of cell-associated human immunodeficiency virus type 1 (HIV-1). For subjects with undetectable HIV-1 RNA levels in plasma, there was a 10,000-fold range of cell-associated virus detected. This method provides a simple and reproducible means for monitoring cell-associated HIV-1.
Identification and Detection of
Stenotrophomonas maltophilia
by rRNA-Directed PCR
Authors: Whitby Paul W. , Carter Karen B. , Burns Jane L. , Royall James A. , LiPuma John J. , Stull Terrence L.
Published: December 2019

Stenotrophomonas maltophilia
has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease,
S. maltophilia
can be cultured from respiratory tract secretions. Identification of
S. maltophilia
can be problematic, and analysis of isolates from the
Burkholderia cepacia
Research Laboratory and Repository showed that several isolates presumptively identified as
B. cepacia
by clinical microbiology laboratories were in fact
S. maltophilia
. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify
S. maltophilia
directly from sputum. The SS-PCR was developed and tested against a panel of 112
S. maltophilia
isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all
S. maltophilia
isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify
S. maltophilia
in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify
S. maltophilia
isolates and which can be used for the direct detection of this organism in CF patient sputum.
Quantitative Analysis of Cytomegalovirus (CMV) Viremia Using the pp65 Antigenemia Assay and the COBAS AMPLICOR CMV MONITOR PCR Test after Blood and Marrow Allogeneic Transplantation
Authors: Boivin G. , Bélanger R. , Delage R. , Béliveau C. , Demers C. , Goyette N. , Roy J.
Published: December 2019

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (
P
= 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.
Antigenic Diversity of
Haemophilus somnus
Lipooligosaccharide: Phase-Variable Accessibility of the Phosphorylcholine Epitope
Authors: Howard Michael D. , Cox Andrew D. , Weiser Jeffrey N. , Schurig Gerhardt G. , Inzana Thomas J.
Published: December 2019

The lipooligosaccharide (LOS) of
Haemophilus somnus
undergoes antigenic phase variation, which may facilitate evasion from the bovine host immune response and/or colonization and dissemination. However, LOS antigenic diversity in
H. somnus
has not been adequately investigated. In this study, monoclonal antibodies (MAbs) specific to various LOS epitopes were used to investigate antigenic variation and stability in LOS from
H. somnus
strains and phase variants. Clinical isolates of
H. somnus
exhibited intrastrain, as well as interstrain, antigenic heterogeneity in LOS when probed with MAbs to outer core oligosaccharide epitopes in an enzyme-linked immunosorbent assay (ELISA). However, epitopes reactive with MAbs directed predominately to the inner core heptose region were highly conserved. At least one epitope, which was expressed in few strains, was identified. One LOS component affected by phase variation was identified as phosphorylcholine (PCho), which is linked to the primary glucose residue. Inhibition ELISA, immunoblotting, and electrospray-mass spectrometry were used to confirm that MAb 5F5.9 recognized PCho. LOS reactivity with MAb 5F5.9 was associated with loss of most of the outer core oligosaccharide, indicating that reactivity with PCho was affected by phase variation of the glycose residues in this region. Our results indicate that outer core epitopes of
H. somnus
LOS exhibit a high degree of random, phase-variable antigenic heterogeneity and that such heterogeneity must be considered in the design of vaccines and diagnostic tests.
Effects of Amplification Facilitators on Diagnostic PCR in the Presence of Blood, Feces, and Meat
Authors: Abu Al-Soud Waleed , Rådström Peter
Published: December 2019

The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing
rTth
or
Taq
DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing
Taq
DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases.
Taq
DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and
rTth
was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of
rTth
containing meat and of
Taq
DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed
Taq
DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.
Genetic Heterogeneity in
Mycobacterium tuberculosis
Isolates Reflected in IS
6110
Restriction Fragment Length Polymorphism Patterns as Low-Intensity Bands
Authors: de Boer Annette S. , Kremer Kristin , Borgdorff Martien W. , de Haas Petra E. W. , Heersma Herre F. , van Soolingen Dick
Published: December 2019

Mycobacterium tuberculosis
isolates with identical IS
6110
restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS
6110
RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple
M. tuberculosis
strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS
6110
-mediated genetic adaptation of
M. tuberculosis
to changes in the environmental conditions during the dormant state or reactivation thereafter.
Strain Identification of
Trichophyton rubrum
by Specific Amplification of Subrepeat Elements in the Ribosomal DNA Nontranscribed Spacer
Authors: Jackson Colin J. , Barton Richard C. , Kelly Steven L. , Evans E. Glyn V.
Published: December 2019

Trichophyton rubrum
is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the
T. rubrum
ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species,
Trichophyton violaceum
,
Trichophyton gourvilii
, and
Trichophyton soudanense
, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing
T. rubrum
will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.
Duodenal Microflora in Very-Low-Birth-Weight Neonates and Relation to Necrotizing Enterocolitis
Authors: Hoy Christine M. , Wood Christopher M. , Hawkey Peter M. , Puntis John W. L.
Published: December 2019

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the neonatal period. Small-bowel overgrowth with aerobic gram-negative bacteria has previously been implicated in the development of NEC. This prospective study performed quantitative bacteriology on 422 duodenal aspirates collected from 122 very-low-birth-weight (<1,500-g) newborns, at the time of routine changing of nasogastric tubes. Isolates of
Enterobacteriaceae
were typed by repetitive extragenic, palindromic PCR and pulsed-field gel electrophoresis. One or more samples from 50% of these infants yielded gram-negative bacteria, predominantly
Escherichia coli
,
Klebsiella
spp., and
Enterobacter
spp., with counts up to 10
8
CFU/g. The proportion of samples with gram-negative bacteria increased with postnatal age, while the percentage of sterile samples declined. Molecular typing revealed marked temporal clustering of indistinguishable strains. All infants had been fed prior to isolation of gram-negative organisms. Antibiotic use had no obvious effect on colonization with
Enterobacteriaceae
. There were 15 episodes of suspected NEC (stage I) and 8 confirmed cases of NEC (2 stage II and 6 stage III) during the study period. Duodenal aspirates were collected prior to clinical onset in 13 episodes of NEC. Seven of these yielded
Enterobacteriaceae
, of which five strains were also isolated from infants without NEC. Very-low-birth-weight infants have high levels of duodenal colonization with
Enterobacteriaceae
, with evidence of considerable cross-colonization with indistinguishable strains. There was no association between duodenal colonization with particular strains of
Enterobacteriaceae
and development of NEC.
Characterization of Chloramphenicol and Florfenicol Resistance in
Escherichia coli
Associated with Bovine Diarrhea
Authors: White David G. , Hudson Charlene , Maurer John J. , Ayers Sherry , Zhao Shaohua , Lee Margie D. , Bolton Lance , Foley Thomas , Sherwood Julie
Published: December 2019

Florfenicol, a veterinary fluorinated analog of thiamphenicol, is approved for treatment of bovine respiratory pathogens in the United States. However, florfenicol resistance has recently emerged among veterinary
Escherichia coli
isolates incriminated in bovine diarrhea. The
flo
gene, which confers resistance to florfenicol and chloramphenicol, has previously been identified in
Photobacterium piscicida
and
Salmonella enterica
serovar Typhimurium DT104. The
flo
gene product is closely related to the CmlA protein identified in
Pseudomonas aeruginosa
. The
cmlA
gene confers nonenzymatic chloramphenicol resistance via an efflux mechanism. Forty-eight
E. coli
isolates recovered from calves with diarrhea, including 41 that were both chloramphenicol and florfenicol resistant, were assayed for the presence of both
flo
and
cmlA
genes. Forty-two of the 44 isolates for which florfenicol MICs were ≥16 μg/ml were positive via PCR for the
flo
gene. All
E. coli
isolates for which florfenicol MICs were ≤8 μg/ml were negative for the
flo
gene (
n
= 4). Twelve
E. coli
isolates were positive for
cmlA
, and chloramphenicol MICs for all 12 were ≥32 μg/ml. Additionally, eight isolates were positive for both
flo
and
cmlA
, and both florfenicol and chloramphenicol MICs for these isolates were ≥64 μg/ml. DNA sequence analysis of the
E. coli flo
gene demonstrated 98% identity to the published GenBank sequences of both serovar Typhimurium

flo
St

and
P. piscicida pp-flo
. The
flo
gene was identified on high-molecular-weight plasmids of approximately 225 kb among the majority of florfenicol-resistant
E. coli
isolates. However, not all of the florfenicol-resistant
E. coli
isolates tested contained the large
flo
-positive plasmids. This suggests that several of the
E. coli
isolates may possess a chromosomal
flo
gene. The
E. coli flo
gene specifies nonenzymatic cross-resistance to both florfenicol and chloramphenicol, and its presence among bovine
E. coli
isolates of diverse genetic backgrounds indicates a distribution much wider than previously thought.
Evaluation of Random Amplified Polymorphic DNA Typing of
Pseudomonas aeruginosa
Authors: Campbell Maureen , Mahenthiralingam Eshwar , Speert David P.
Published: December 2019

A method for distinguishing among
Pseudomonas aeruginosa
strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.
Antimicrobial Resistance ofSalmonellaIsolates from Swine
Authors: Gebreyes Wondwossen A. , Davies Peter R. , Morrow W. E. Morgan , Funk Julie A. , Altier Craig
Published: December 2019

We examined the antimicrobial resistance of 1,257 isolates of 30 serovars ofSalmonella entericasubsp.entericaisolated from swine. Serovars Typhimurium and Typhimurium var. Copenhagen were widespread and were frequently multidrug resistant, with distinct resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline and to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, respectively.
High-Pathogenicity Island of
Yersinia
spp. in
Escherichia coli
Strains Isolated from Diarrhea Patients in China
Authors: Xu Jian-guo , Cheng Bokun , Wen Xuanqin , Cui Shuyu , Ye Changyun
Published: December 2019

The high-pathogenicity island (HPI) of
Yersinia
has been observed in 93% of 60 enteroadhesive
Escherichia coli
strains and 80% of
E. coli
strains isolated from blood samples. In the present study we investigated 671 fecal samples from patients with diarrhea in Shandong Province, China, and isolated HPI-harboring
E. coli
from 6.26% of the samples. The isolation rates for patients with diarrhea in three age groups, 10 to 20, 30 to 40, and 50 to 60 years, were 6.70, 12.35, and 10.81%, respectively. Therefore, HPI-harboring
E. coli
is the third most frequently isolated enteric pathogen from patients with diarrhea. Vomiting and abdominal pain were recorded for 33.33 and 66.67% of the patients, respectively. Stools with blood were observed for 9.52% of the patients. Twenty-four of 42 (57%) patients experienced a temperature over 37.4°C. These observations indicate that HPI-harboring
E. coli
is one of the major causes of diarrheal disease in China and that the clinical symptoms caused by HPI-harboring
E. coli
differ from those caused by enteroadhesive
E. coli.
Simplified Hepatitis C Virus Genotyping by Heteroduplex Mobility Analysis
Authors: White Peter A. , Zhai Xinyan , Carter Ian , Zhao Yue , Rawlinson William D.
Published: December 2019

Heteroduplex mobility analysis (HMA) was used to genotype hepatitis C viruses (HCV) with PCR fragments derived from the 5′ untranslated region (5′-UTR) or the NS5b region. HCV 5′-UTR fragments were amplified from 296 serum samples by use of a combined reverse transcription-PCR assay, and the genotypes of isolates were determined by sequencing. HCV genotype distributions in Australia were 39% for genotype 1a, 15% for 1b, 3% for 1a/b, <1% for 2a/c, 5% for 2b, 34% for 3a, <1% for 3b, and 1% for 4, and 1% of patients were infected with more than one genotype. Pairwise HMA of subtypes 1a, 1b, 2a/c, 2b, 3a, 3b, 4a, and 6a demonstrated that five distinct heteroduplex patterns were formed between the eight subtypes. A reference panel that contained a representative of each pattern (1a, 2b, 3a, 4a, and 6a) was used for genotyping. The pattern of heteroduplexes formed when a test isolate was mixed with the five reference isolates was correlated with the genotype, as determined by sequencing. Genotypes determined by HMA correlated exactly with sequencing results within the groups 1, 2, 3a, 3b/4, and 6. HMA was also used to simplify the identification of mixed infection with two HCV genotypes. In further studies, with amplicons from the NS5b region, HMA classified isolates into their respective subtypes, and the heteroduplex mobility ratio correlated closely with nucleotide sequence variation at the isolate, subtype, and genotype levels. HMA provides an adaptable, inexpensive, and rapid method of genotyping HCV that requires fewer resources than DNA sequencing.
Sequence Analysis and Clinical Significance of the
iceA
Gene from
Helicobacter pylori
Strains in Japan
Authors: Ito Yoshiyuki , Azuma Takeshi , Ito Shigeji , Suto Hiroyuki , Miyaji Hideki , Yamazaki Yukinao , Kato Takuji , Kohli Yoshihiro , Keida Yoshihide , Kuriyama Masaru
Published: December 2019

The
Helicobacter pylori iceA
gene was recently identified as a genetic marker for the development of peptic ulcer in a Western population. To assess the significance of
iceA
subtypes of
H. pylori
in relation to peptic ulcer, 140 Japanese clinical isolates (88 from Fukui and 52 from Okinawa) were characterized. Sequence analysis of the
iceA1
gene from 25 representative Japanese strains was also carried out to identify the differences in
iceA
between the ulcer group and the gastritis group. The
iceA1
genotype was not correlated with the presence of peptic ulceration in either area. In addition, sequence analysis led to identification of five deletions and five point mutations (a nonsense mutation or a 1-bp insertion) within the
iceA1
open reading frame corresponding to previously published sequences. These mutations were identified in both clinical groups (ulcer and gastritis groups) in each area. Local DNA sequence analysis revealed that the endpoints of all five deletions coincided with direct repeats. We also found four strains that carried longer
iceA1
open reading frames compared with that for strain 60190. In conclusion, carriage of an
iceA1
strain does not seem to be a risk factor for peptic ulcer in Japanese subjects. The critical mutations in the
iceA1
gene in some isolates from patients with peptic ulcers suggested that IceA does not participate in the pathogenesis of peptic ulcer in Japan. We also found deletion hot spots that were associated with direct repeats in
iceA1
and that favored a small-deletion model of slipped mispairing events during replication. We showed that
iceA1
sequence variations may be useful tools for analysis of the population genetics of
H. pylori
.
Antimicrobial Resistance of
Neisseria gonorrhoeae
and High Prevalence of Ciprofloxacin-Resistant Isolates in Japan, 1993 to 1998
Authors: Tanaka Masatoshi , Nakayama Hiroshi , Haraoka Masashi , Saika Takeshi , Kobayashi Intetsu , Naito Seiji
Published: December 2019

To assess the antimicrobial resistance of
Neisseria gonorrhoeae
isolated from 1993 through 1998 in Japan, susceptibility testing was conducted on 502 isolates. Selected isolates were characterized by auxotype and analysis for mutations within the quinolone resistance-determining region (QRDR) in the
gyrA
and
parC
genes, which confer fluoroquinolone resistance on the organism. Plasmid-mediated penicillin resistance (penicillinase-producing
N. gonorrhoeae
) decreased significantly from 1993–1994 (7.9%) to 1997–1998 (2.0%). Chromosomally mediated penicillin resistance decreased from 1993–1994 (12.6%) to 1995–1996 (1.9%) and then increased in 1997–1998 (10.7%). Chromosomally mediated tetracycline resistance decreased from 1993–1994 (3.3%) to 1997–1998 (2.0%), and no plasmid-mediated high-level tetracycline resistance was found. Isolates with ciprofloxacin resistance (MIC ≥ 1 μg/ml) increased significantly from 1993–1994 (6.6%) to 1997–1998 (24.4%). The proline-requiring isolates were less susceptible to ciprofloxacin than the prototrophic or arginine-requiring isolates. Ciprofloxacin-resistant isolates contained three or four amino acid substitutions within the QRDR in the GyrA and ParC proteins.
Molecular Detection and Epidemiology of Sapporo-Like Viruses
Authors: Vinjé Jan , Deijl Hanneke , van der Heide Reina , Lewis David , Hedlund Kjell-Olof , Svensson Lennart , Koopmans Marion P. G.
Published: December 2019

Sapporo-like viruses (SLVs) are associated with acute gastroenteritis in humans. Due to a limited supply of available reagents for diagnosis, little is known about the incidence and pathogenicity of these viruses. We have developed a first-generation generic reverse transcriptase (RT) PCR assay based on a single primer pair targeting the RNA polymerase gene. With this assay, 55 (93%) of the 59 stool specimens collected in a 10-year period of time (1988 to 1998) and containing typical caliciviruses by electron microscopy tested positive and could be confirmed by Southern hybridization. By phylogenetic analysis, most SLV strains could be classified into one of the three recently described genotypes. However, three samples clustered separately, forming a potential new genotype. We sequenced the complete capsid gene of one of the strains in this cluster: Hu/SLV/Stockholm/97/SE. Alignment of the capsid sequences showed 40 to 74% amino acid identity among strains of the different clusters. Phylogenetic analysis of the aligned sequences confirmed the placing of Hu/SLV/Stockholm/97/SE into a new distinct genetic cluster. This is the first report on the development of a broadly reactive RT-PCR assay for the detection of SLVs.
Screening for Hepatitis C Virus in Human Immunodeficiency Virus-Infected Individuals
Authors: Thio Chloe L. , Nolt Karen R. , Astemborski Jacquie , Vlahov David , Nelson Kenrad E. , Thomas David L.
Published: December 2019

Immunosuppression from human immunodeficiency virus (HIV) may impair antibody formation, and false-negative hepatitis C virus antibody (anti-HCV) tests have been reported in individuals coinfected with HIV and HCV. It is unknown if the frequency of false-negative tests is sufficiently high to change screening recommendations in this setting. Thus, the prevalence of false-negative results for anti-HCV by third-generation tests was determined with samples from HIV-infected individuals. Sera from 559 HIV-infected and 944 HIV-negative prospectively followed injection drug users were tested for anti-HCV by a third-generation enzyme immunoassay and for HCV RNA by using a branched DNA assay and the HCV COBAS AMPLICOR system. Of 559 HIV-infected participants, 547 (97.8%) were anti-HCV positive. One of the remaining 12 anti-HCV-negative participants was HCV RNA positive, and she later developed detectable anti-HCV. Of the 944 HIV-negative participants, 825 (87.4%) were anti-HCV positive. One of the remaining 119 anti-HCV-negative participants was HCV RNA positive, and she also developed detectable anti-HCV at a later visit. These data indicate that HIV infection does not alter the approach to hepatitis C virus screening, which should be performed with third-generation assays for anti-HCV unless acute infection is suspected.
Evaluation of a Specific Nested PCR Targeting Domain III of the 23S rRNA Gene of “
Tropheryma whippelii
” and Proposal of a Classification System for Its Molecular Variants
Authors: Hinrikson Hans Peter , Dutly Fabrizio , Altwegg Martin
Published: December 2019

“
Tropheryma whippelii
”-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of “
T. whippelii
” are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that “
T. whippelii
” DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a “
T. whippelii
”-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain “
T. whippelii
” 16S rDNA. All of these specimens were also positive for “
T. whippelii
” 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of “
T. whippelii
” DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial “
T. whippelii
” 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of “
T. whippelii
.” This preliminary scheme may provide a basis for further epidemiological and clinical studies with “
T. whippelii
” and associated diseases.
Serology of Culture-Confirmed Cases of Human Granulocytic Ehrlichiosis
Authors: Aguero-Rosenfeld Maria E. , Kalantarpour Fatemeh , Baluch Mehdi , Horowitz Harold W. , McKenna Donna F. , Raffalli John T. , Hsieh Tze-chen , Wu Joseph , Dumler J. Stephen , Wormser Gary P.
Published: December 2019

We evaluated the antibody responses in the sera of 24 patients with culture-confirmed human granulocytic ehrlichiosis (HGE). Antibody titers were measured by an indirect immunofluorescent-antibody assay (IFA) by using a local human isolate as the source of antigen. All patients received appropriate antimicrobial treatment. One hundred five serum specimens collected at baseline and at periodic intervals for up to 14 months were included in the study. Seroconversion was observed in 21 of 23 patients (91.3%) from whom convalescent-phase sera were obtained. Antibodies were first detected at an average of 11.5 days after onset of symptoms. Peak titers (≥2,560 for 71.4% of patients and ≥640 for 95.2% of patients) were obtained an average of 14.7 days after onset of symptoms. Eleven of 13 patients (84.6%) from whom sera were collected between 6 and 10 months after onset of symptoms were still seropositive, and sera from 5 of 10 (50%) patients tested positive between 11 and 14 months after onset of symptoms. For a subset of 71 serum specimens from 17 patients with culture-confirmed HGE also tested by IFA by using either a human isolate from Wisconsin or an
Ehrlichia equi
isolate from a horse, there was qualitative agreement for 62 serum specimens (87.3%). Peak titers were higher, however, with the local human HGE isolate, but the difference was not statistically significant. In summary, most patients with culture-confirmed HGE develop antibodies within 2 weeks of onset of symptoms. Antibodies reach high titers during the first month and remain detectable in about one-half of patients at 1 year after onset of symptoms.
Prevalence of GB Virus C/Hepatitis G Virus Infection among Various Populations in Surabaya, Indonesia, and Identification of Novel Groups of Sequence Variants
Authors: Handajani Retno , Soetjipto  , Lusida Maria Inge , Suryohudoyo Purnomo , Adi Pangestu , Setiawan Poernomo Boedi , Nidom Chairul Anwar , Soemarto R. , Katayama Yuko , Fujii Motoko , Hotta Hak
Published: December 2019

A molecular epidemiological study was performed to investigate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among various populations in Surabaya, Indonesia. The prevalence of GBV-C/HGV RNA, determined by reverse transcription-PCR for a portion of the NS3 region of the viral genome, was 2.7% (4 of 150) among randomly collected blood donor sera, which were all negative for both hepatitis B virus surface antigen and antibodies against hepatitis C virus (HCV). On the other hand, the prevalence among anti-HCV-positive blood donors was 17.8% (13 of 73), with the ratio being significantly higher than that observed with the anti-HCV-negative blood donors (
P
< 0.001). A high prevalence of GBV-C/HGV infection was also observed among patients with chronic liver disease, such as chronic hepatitis (5.7%), liver cirrhosis (11.5%), and hepatocellular carcinoma (7.0%), and patients on maintenance hemodialysis (29.0%). No correlation was observed between GBV-C/HGV viremia and serum alanine aminotransferase levels in the populations tested, suggesting the possibility that GBV-C/HGV does not cause apparent liver injury. Phylogenetic analysis of sequences of a portion of the 5′ untranslated region and the E1 region of the viral genome identified, in addition to a previously reported then novel group of GBV-C/HGV variants (group 4), another novel group of variants (group 5). This result suggests that GBV-C/HGV can be classified into at least five genetic groups. GBV-C/HGV isolates of group 4 and group 5 were each shown to comprise approximately 40% of the total Indonesian isolates.
A Novel and Rapid PCR-Based Method for Genotyping Human Papillomaviruses in Clinical Samples
Authors: Nelson Joshua H. , Hawkins Gregory A. , Edlund Karin , Evander Magnus , Kjellberg Lennart , Wadell Göran , Dillner Joakim , Gerasimova Tsilya , Coker Ann L. , Pirisi Lucia , Petereit Daniel , Lambert Paul F.
Published: December 2019

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5
+
-GP6
+
consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil
N
-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a “T-sequencing ladder” was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.
Use of PCR To Detect
Leishmania
(
Viannia
) spp. in Dog Blood and Bone Marrow
Authors: Reithinger Richard , Lambson Bronwen E. , Barker Douglas C. , Davies Clive R.
Published: December 2019

A PCR-based protocol for the detection of
Leishmania
(
Viannia
) parasites in canine blood, buffy coat, and bone marrow was developed and was then tested with field samples taken from a random sample of 545 dogs from villages in Peru where
Leishmania
(
Viannia
)
braziliensis
and
Leishmania
(
Viannia
)
peruviana
are endemic. Comparative tests with cultured parasites mixed with dog blood showed that the PCR assay's sensitivity was significantly dependent on the DNA extraction protocol and the PCR primers used. Mass screening of field samples by the preferred PCR protocol detected American cutaneous leishmaniasis (ACL) in 44 of 545 (8.1%) dogs; 31 of 402 (7.7%), 20 of 223 (9.0%), and 8 of 46 (17.4%) were PCR positive when whole blood, buffy coat, and bone marrow aspirates, respectively, were tested. The high prevalence of
Leishmania
in both asymptomatic (7.6%) and symptomatic (18.0%) dogs provides further circumstantial evidence for their suspected role as reservoir hosts of ACL and indicates that hematogenous dissemination of parasites may be a more common pathological phenomenon than has previously been acknowledged. However, unlike for zoonotic visceral leishmaniasis, the comparatively low prevalence of
Leishmania
(
Viannia
) in the blood of symptomatic dogs indicates that PCR with blood cannot be the “gold standard” for the (mass) screening of samples in epidemiological studies.
Comparison of PCR, Antigenemia Assay, and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation
Authors: Weinberg Adriana , Hodges Tony N. , Li Shaobing , Cai Guanyung , Zamora Martin R.
Published: December 2019

The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.
Analysis of Genetic Variability within the Immunodominant Epitopes of Envelope gp41 from Human Immunodeficiency Virus Type 1 (HIV-1) Group M and Its Impact on HIV-1 Antibody Detection
Authors: Dorn Jonathan , Masciotra Silvina , Yang Chunfu , Downing Robert , Biryahwaho Benon , Mastro Timothy D. , Nkengasong John , Pieniazek Danuta , Rayfield Mark A. , Hu Dale J. , Lal Renu B.
Published: December 2019

The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n= 42), B (n= 62), B′ (n= 13), C (n= 38), D (n= 41), E (n= 18), F (n= 27), and G (n= 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B′, in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n= 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.