bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide-Based OspC Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Lyme Borreliosis
Authors: Mathiesen Marianne J. , Christiansen Michael , Hansen Klaus , Holm Arne , Åsbrink Eva , Theisen Michael
Published: December 2019

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of
Borrelia burgdorferi
. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (≤8%) displayed IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the
B. burgdorferi
flagellum (Fla). The sensitivity of the IgM pepC10 ELISA was slightly lower (P < 0.04) than that of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a significantly better quantitative discrimination between seropositive patients with NB and controls (P < 0.008). The specificity of the pepC10 ELISA was similar to those of the rOspC ELISA and the Fla ELISA for relevant controls including patients with syphilis and mononucleosis. Although the overall diagnostic sensitivity of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB patients, respectively, were antibody positive only by the pepC10 ELISA. Thus, use of a diagnostic test for LB based on the detection of IgM antibodies to pepC10 and Fla has increased sensitivity for the diagnosis of early LB.
16S Ribosomal DNA Typing for Identification of Pathogens in Patients with Bacterial Keratitis
Authors: Knox C. Michele , Cevellos Vickey , Dean Deborah
Published: December 2019

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.
Evaluation of Hemagglutinin Protein-Specific Immunoglobulin M for Diagnosis of Measles by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Protein Produced in a High-Efficiency Mammalian Expression System
Authors: Bouche Fabienne B. , Brons Nicolaas H. C. , Houard Sophie , Schneider Francois , Muller Claude P.
Published: December 2019

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.
Differentiation between
Streptococcus gallolyticus
Strains of Human Clinical and Veterinary Origins and
Streptococcus bovis
Strains from the Intestinal Tracts of Ruminants
Authors: Devriese Luc A. , Vandamme Peter , Pot Bruno , Vanrobaeys Mia , Kersters Karel , Haesebrouck Freddy
Published: December 2019

Strains formerly identified as
Streptococcus bovis
were allotted to two groups by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell proteins. Strains from humans with infections, mostly patients with endocarditis, and strains from pigeons with septicemia clustered with the recently described species
Streptococcus gallolyticus
. The original
S. bovis
type strain and strains exclusively from ruminants formed the second cluster. The findings indicate that
S. gallolyticus
is more likely to be involved in human and animal infections than
S. bovis
. Growth characteristics and several biochemical reactions were found to be useful in the differentiation of
S. gallolyticus
from
S. bovis.
Rubella Virus and Chronic Joint Disease: Is There an Association?
Authors: Bosma Trent J. , Etherington John , O’Shea Siobhan , Corbett Karen , Cottam Fiona , Holt Lennox , Banatvala J. E. , Best Jennifer M.
Published: December 2019

Synovial fluid samples and/or biopsies from 79 patients with various chronic inflammatory joint diseases or traumatic joint injury were tested for rubella virus (RV) in order to confirm or refute results from other studies that suggested RV as a cause of chronic inflammatory joint disease. Sixty-eight of the 72 patients tested had RV antibodies. RV RNA was detected by reverse transcription-PCR in the synovial fluid cells from two patients. RV was also isolated by cell culture from the synovial fluid of one of these two patients. This patient was a 42-year-old female with common variable immune deficiency and
Mycoplasma hominis
arthritis, while the other was a 68-year-old female with rheumatoid arthritis. While these results fail to confirm that RV is associated with chronic inflammatory joint disease, they suggest that RV may persist within a joint and be reactivated when cell-mediated immunity is suppressed.
Genetic Variability among Group A and Group B Respiratory Syncytial Viruses in a Children’s Hospital
Authors: Coggins Wanicha Buraphacheep , Lefkowitz Elliot J. , Sullender Wayne M.
Published: December 2019

Respiratory syncytial (RS) viruses isolated over three epidemic periods in a children’s hospital in the United States were analyzed. The viruses (
n
= 174) were characterized as to major antigenic group (group A or B) by a PCR-based assay. Group A RS viruses were dominant the first 2 years, followed by a year with group B dominance (ratios of group A to group B viruses for epidemic periods, 56/4 for 1993–1994, 42/3 for 1994–1995, and 19/50 for 1995–1996). Genetic variability within the groups was assessed by restriction fragment analysis of PCR products; 79 isolates were also analyzed by nucleotide sequence determination of a variable region of the glycoprotein G gene. Among the group A RS virus isolates, this G-protein variable region had amino acid differences of as great as 38%. The G-protein amino acids of the group A viruses differed by up to 31% from the G-protein amino acids of a prototype (A2) group A virus. Among the group B RS virus G proteins, amino acid differences were as great as 14%. The G-protein amino acids of the group B viruses differed by up to 27% from the G-protein amino acids of a prototype (18537) group B virus. The group A and group B RS viruses demonstrated genetic variability between years and within individual years. Phylogenetic analysis revealed that there were multiple evolutionary lineages among both the group A and group B viruses. Among the recent group B isolates, variability was less than that seen for the group A viruses. However, comparisons to prototype strains revealed that the group B RS viruses may vary more extensively than was observed over the 3 years studied in the present investigation.
Standardization of the Human Cytomegalovirus Antigenemia Assay by Means of In Vitro-Generated pp65-Positive Peripheral Blood Polymorphonuclear Leukocytes
Authors: Gerna Giuseppe , Percivalle Elena , Torsellini Maria , Revello M. Grazia
Published: December 2019

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at −80°C, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.
Adherence Patterns and Adherence-Related DNA Sequences in
Escherichia coli
Isolates from Children with and without Diarrhea in São Paulo City, Brazil
Authors: Gomes Tânia A. T. , Vieira Mônica A. M. , Abe Cecilia M. , Rodrigues Daleth , Griffin Patricia M. , Ramos Sônia R. T. S.
Published: December 2019

The correlation between various adherence patterns and adherence-related DNA sequences in
Escherichia coli
isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent
E. coli
did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related
daaC
probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.
Molecular Epidemiology of Ampicillin-Resistant Non-β-Lactamase-Producing
Haemophilus influenzae
Authors: Gazagne Louise , Delmas Catherine , Bingen Edouard , Dabernat Henri
Published: December 2019

Resistance to ampicillin without β-lactamase production is not a frequent occurrence among
Haemophilus influenzae
strains. This kind of resistance is encountered in unencapsulated strains isolated from bronchial secretions and ear, nose, and throat specimens and is exceptional in
H. influenzae
type b. We studied 29 of these strains from various areas in France and 2 reference strains. Strains were compared by using ribotyping, arbitarily primed PCR with two primers, and pulsed-field gel electrophoresis. Each technique enabled the identification of 20 to 23 different patterns among the 31 strains. The combination of the different patterns for the strains obtained by the different techniques provided 27 distinct profiles. According to these results, it seems that the clonal propagation of these resistant strains does not occur.
Sequence Variation within Three Important Cytomegalovirus Gene Regions in Isolates from Four Different Patient Populations
Authors: Zweygberg Wirgart Benita , Brytting Maria , Linde Annika , Wahren Britta , Grillner Lena
Published: December 2019

We determined the nucleotide (nt) and amino acid (aa) heterogeneities of three distinct regions of the human cytomegalovirus (CMV) genome for 46 low-passage CMV isolates from four different patient populations (congenitally infected infants, children attending day-care centers, renal transplant recipients, and human immunodeficiency virus-infected individuals) and for two laboratory strains (CMV Ad169 and Towne). The gene regions for the major immediate-early (MIE) exon 4 gene (nt positions 1702 to 1982, aa positions 152 to 244), the DNA polymerase gene (nt positions 2797 to 3046, aa positions 713 to 795), and the glycoprotein B (gB) gene (nt positions 1698 to 1884, aa positions 567 to 628) were sequenced. The sequence information was used to design sets of nested PCR primers directed against the most highly conserved regions identified. MIE was the most variable gene region compared to the variability of the DNA polymerase and gB gene regions. Comparison of the sequences of all 46 isolates with that of Ad169 revealed nt and aa sequence homologies of 87.9 and 87.2%, respectively, within the MIE gene compared to 92.8 and 100% homologies, respectively, within the DNA polymerase gene and 93 and 95.2% homologies, respectively, within the gB gene. Within the MIE gene, compared to the Ad169 nt sequence the homology at the nt level among isolates obtained from children attending day-care centers was high (96.4%), while it was lower (90%) among isolates obtained from the other three patient populations. Preliminary results of a nested PCR with oligonucleotide primers selected from the DNA polymerase gene region with a low level of nt sequence variation indicates that primers selected from this region might be more powerful for use in PCR than primers selected from the MIE gene region.
Disseminated Aspergillosis Caused by
Aspergillus ustus
in a Patient Following Allogeneic Peripheral Stem Cell Transplantation
Authors: Iwen Peter C. , Rupp Mark E. , Bishop Michael R. , Rinaldi Michael G. , Sutton Deanna A. , Tarantolo Stefano , Hinrichs Steven H.
Published: December 2019

The first case of disseminated aspergillosis caused by
Aspergillus ustus
in an allogeneic peripheral stem cell transplant patient is described. The patient, a 46-year-old female with a history of myelodysplastic syndrome, underwent high-dose chemotherapy and total body irradiation prior to transplantation. She was released from the hospital 49 days posttransplant (p.t.) in a stable condition with an absolute neutrophil count (ANC) of 2,700 cells per μl. Multiple antimicrobial agents, including itraconazole (ITR), were prescribed during hospitalization and at the time of discharge. Three days after discharge, the patient was readmitted with hemorrhagic cystitis, persistent thrombocytopenia, and bilateral pulmonary consolidation, although no fever was present. The ANC at the time of readmission was 3,500. Upon detection of a pulmonary nodule (day 67 p.t.), a bronchoalveolar lavage was performed; the lavage fluid was positive for both cytomegalovirus and parainfluenza virus and negative for fungus. The patient was placed on ganciclovir. A biopsy specimen from a leg lesion also noted on day 67 p.t. revealed septate hyphae consistent with
Aspergillus
species, and a culture subsequently yielded
Aspergillus ustus
. Confirmation detection of
A. ustus
was made by demonstration of characteristic reproductive structures with the presence of Hülle cells. On day 67 p.t., ITR was discontinued and liposomal amphotericin B (AMB) was initiated. The patient’s condition worsened, and she died 79 days p.t. At the time of autopsy, septate hyphae were present in heart, thyroid, and lung tissues, with lung tissue culture positive for
A. ustus
. In vitro susceptibility testing indicated probable resistance to AMB but not to ITR. This case supports the need for the development of rapid methods to determine antifungal susceptibility.
Mink Lung Epithelial Cells: Unique Cell Line That Supports Influenza A and B Virus Replication
Authors: Schultz-Cherry Stacey , Dybdahl-Sissoko Naomi , McGregor Martha , Hinshaw Virginia S.
Published: December 2019

We have demonstrated for the first time that a mink lung epithelial cell line (Mv1Lu) supports the replication of influenza A and B viruses, including the recently isolated H5N1 avian and human Hong Kong strains, to titers comparable to those in MDCK cells. These results suggest that Mv1Lu cells might serve as an alternative system for the isolation and cultivation of influenza A and B viruses and may be useful for vaccine development.
Effectiveness of Resins in Neutralizing Antibiotic Activities in Bactec Plus Aerobic/F Culture Medium
Authors: Spaargaren J. , van Boven C. P. A. , Voorn G. P.
Published: December 2019

Incorporating resins in blood culture media can effectively reduce the activities of several antibiotics. It was shown that the activities of some generally used antibiotics decreased by 80 to 90% within 2 h in Bactec Plus Aerobic/F resin-containing culture medium. Bactec vials containing resins were still found to be positive for bacteria when antibiotics were present. The addition of β-lactamase shortened the detection time irrespective of the presence of resins.
Application of Subtype-Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses
Authors: Ueda Minoru , Maeda Akiko , Nakagawa Naoko , Kase Tetsuo , Kubota Ritsuko , Takakura Hikaru , Ohshima Atsushi , Okuno Yoshinobu
Published: December 2019

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase-antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination-inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.
Serotype-Specific Identification of Polioviruses by PCR Using Primers Containing Mixed-Base or Deoxyinosine Residues at Positions of Codon Degeneracy
Authors: Kilpatrick David R. , Nottay Baldev , Yang Chen-Fu , Yang Su-Ju , Da Silva Edson , Peñaranda Silvia , Pallansch Mark , Kew Olen
Published: December 2019

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.
Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay
Authors: Krajden Mel , Comanor Lorraine , Rifkin Oretta , Grigoriew Anna , Minor James M. , Kapke Gordon F.
Published: December 2019

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log
10
HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C.
Rapid Detection of
Candida albicans
in Clinical Samples by DNA Amplification of Common Regions from
C. albicans
-Secreted Aspartic Proteinase Genes
Authors: Flahaut M. , Sanglard D. , Monod M. , Bille J. , Rossier M.
Published: December 2019

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the
Candida albicans
-secreted aspartic proteinase (
SAP
) genes, a multiple-gene family of at least seven members in
C. albicans
. A single pair of primers was designed in order to detect six of these
SAP
genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an
SAP
-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted
Candida
cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (
n
= 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.
Impact of Strain Heterogeneity on Lyme Disease Serology in Europe: Comparison of Enzyme-Linked Immunosorbent Assays Using Different Species of
Borrelia burgdorferi
Sensu Lato
Authors: Hauser Ulrike , Krahl Heide , Peters Helmut , Fingerle Volker , Wilske Bettina
Published: December 2019

For the standardization of serological tests for Lyme borreliosis (LB) in Europe, the influence of the heterogeneity of
Borrelia burgdorferi
sensu lato must be assessed in detail. For this study four immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) with octyl-β-
d
-glucopyranoside extracts of strains PKo (
Borrelia afzelii
), PBi (
Borrelia garinii
), and PKa2 and B31 (both
B. burgdorferi
sensu stricto) were compared. Strains PKo, PBi, and PKa2 at the passages used for antigen preparations abundantly expressed outer surface protein C (OspC), whereas strain B31 at the passage used for antigen preparation did not express OspC. Sera (all from Germany) from 222 patients with clinically defined LB of all stages, 133 blood donors, and 458 forest workers were tested. None of the forest workers had symptoms consistent with LB at the time that the samples were collected. For IgM tests, receiver operating characteristic curves demonstrated that discrimination between sera from patients and blood donors was best with strain PKo and worst with strain B31. The discriminatory abilities of the four IgG ELISAs were similar in a diagnostically reasonable specificity range (90 to 100%). More than 20% of the sera from forest workers reacted strongly in the PKo IgG ELISA (optical density value, >1.5; other assays, less than 8%). Western blots of the sera with the most discrepant ELISA results revealed almost exclusive reactivity with p17. This highly immunogenic antigen is only expressed by strain PKo. This observation might be important for the development of assays enabling discrimination between asymptomatic or previous infection and active disease.
Hepatitis G Virus Infection in Amerindians and Other Venezuelan High-Risk Groups
Authors: Pujol Flor H. , Khudyakov Yury E. , Devesa Marisol , Cong Mian-E. , Loureiro Carmen L. , Blitz Linda , Capriles Freya , Beker Simón , Liprandi Ferdinando , Fields Howard A.
Published: December 2019

Recently, a new virus related to flaviviruses, the hepatitis G virus (HGV), or GBV-C virus, was discovered as a putative blood-borne human pathogen. HGV RNA (NS5 region) was amplified by reverse transcription-nested PCR in the sera of 6 of 64 (9%) hemodialysis patients; 2 of 80 (2.5%) West Yukpa Amerindians, a population with a high rate of HBV infection but negative for HCV infection; and 1 patient with an acute episode of non-A, non-B, non-C hepatitis (NABCH). The patterns of single-strand conformation polymorphism of the amplified products were unique among different specimens and similar on follow-up for hemodialysis patients. All patients tested remained HGV RNA positive 1 and 2 years later, without major sequence variation, except for the NABCH patient, for whom a double infection and an apparent clearance of the original dominant variant was observed after 2 years. The sequences of the NS5 amplified products demonstrated 85 to 90% identity with other reported HGV sequences.
Pooling Urine Samples for Ligase Chain Reaction Screening for Genital
Chlamydia trachomatis
Infection in Asymptomatic Women
Authors: Kacena Katherine A. , Quinn Sean B. , Howell M. René , Madico Guillermo E. , Quinn Thomas C. , Gaydos Charlotte A.
Published: December 2019

The accuracy of pooling urine samples for the detection of genital
Chlamydia trachomatis
infection by ligase chain reaction (LCR) was examined. A model was also developed to determine the number of samples to be pooled for optimal cost savings at various population prevalences. Estimated costs included technician time, laboratory consumables, and assay costs of testing pooled samples and retesting individual specimens from presumptive positive pools. Estimation of population prevalence based on the pooled LCR results was also applied. After individual urine specimens were processed, 568 specimens were pooled by 4 into 142 pools and another 520 specimens were pooled by 10 into 52 pools. For comparison, all 1,088 urine specimens were tested individually. The sample-to-cut-off ratio was lowered from 1.0 to 0.2 for pooled samples, after a pilot study which tested 148 samples pooled by 4 was conducted. The pooling algorithm was 100% (48 of 48) sensitive when samples were pooled by 4 and 98.4% (61 of 62) sensitive when samples were pooled by 10. Although 2.0% (2 of 99) of the negative pools of 4 and 7.1% (1 of 14) of the negative pools of 10 tested presumptive positive, all samples in these presumptive-positive pools were negative when retested individually, making the pooling algorithm 100% specific. In a population with 8% genital
C. trachomatis
prevalence, pooling by four would reduce costs by 39%. The model demonstrated that with a lower prevalence of 2%, pooling eight samples would reduce costs by 59%. Pooling urine samples for detection of
C. trachomatis
by LCR is sensitive, specific, and cost saving compared to testing individual samples.
Stability of Human Immunodeficiency Virus RNA in Blood Specimens as Measured by a Commercial PCR-Based Assay
Authors: Sebire Kimberley , McGavin Kate , Land Sally , Middleton Tracey , Birch Chris
Published: December 2019

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log
10
(approximately threefold) in blood and plasma samples held at room temperature or 4°C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at −70°C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.
Comparative Study of Bacteremias Caused by
Enterococcus
spp. with and without High-Level Resistance to Gentamicin
Authors: Caballero-Granado Francisco Javier , Cisneros J. M. , Luque R. , Torres-Tortosa M. , Gamboa F. , Díez F. , Villanueva J. L. , Pérez-Cano R. , Pasquau J. , Merino D. , Menchero A. , Mora D. , López-Ruz M. A. , Vergara A. , Infecciosas for the Grupo Andaluz Para El Estudio De Las Enfermedades
Published: December 2019

A prospective, multicenter study was carried out over a period of 10 months. All patients with clinically significant bacteremia caused by
Enterococcus
spp. were included. The epidemiological, microbiological, clinical, and prognostic features and the relationship of these features to the presence of high-level resistance to gentamicin (HLRG) were studied. Ninety-three patients with enterococcal bacteremia were included, and 31 of these cases were caused by HLRG (33%). The multivariate analysis selected chronic renal failure, intensive care unit stay, previous use of antimicrobial agents, and
Enterococcus faecalis
species as the independent risk factors that influenced the development of HLRG. The strains with HLRG showed lower levels of susceptibility to penicillin and ciprofloxacin. Clinical features (except for chronic renal failure) were similar in both groups of patients. HLRG did not influence the prognosis for patients with enterococcal bacteremia in terms of either the crude mortality rate (29% for patients with bacteremia caused by enterococci with HLRG and 28% for patients not infected with strains with HLRG) or the hospital stay after the acquisition of enterococcal bacteremia. Hemodynamic compromise, inappropriate antimicrobial therapy, and mechanical ventilation were revealed in the multivariate analysis to be the independent risk factors for mortality. Prolonged hospitalization was associated with the nosocomial acquisition of bacteremia and polymicrobial infections.
Evaluation of the RapID CB Plus System for Identification of
Corynebacterium
Species and Other Gram-Positive Rods
Authors: Hudspeth Marie K. , Gerardo Sharon Hunt , Citron Diane M. , Goldstein Ellie J. C.
Published: December 2019

Due to the difficulty of identifying
Corynebacterium
spp. with standard methods, we compared them with the RapID CB Plus system (Remel, Lenexa, Kans. [formerly Innovative Diagnostic Systems, Norcross, Ga.]), which consists of 4 carbohydrate and 14 preformed enzyme tests, for the identification of 98 clinical isolates of
Corynebacterium
sp., other coryneforms,
Listeria monocytogenes
, and 17 ATCC strains. Forty (95%) of 42 strains of
Corynebacterium
spp. were accurately identified to the species level by the RapID CB Plus system, and two additional strains of
C. striatum
were identified with one additional conventional test for lipid requirement. Twenty-seven (75%) of the 36 coryneform strains tested were identified correctly to the species level. However, three of four strains of
Brevibacterium
sp. and all seven of the
L. monocytogenes
strains were identified to the genus level only.
Actinomyces
strains had variable results, and the one strain of
Arcanobacterium haemolyticum
tested was not identified. Overall, the RapID CB Plus system compared favorably with the conventional methods, was easy to inoculate and interpret, and is promising as a new method for identification of gram-positive bacilli.
Genetic Heterogeneity of Strains of
Neisseria meningitidis
Belonging to Serotype 22 Isolated in the Czech Republic
Authors: Musilek Martin , Giorgini Dario , Hamadouche Nora , Kriz Paula , Taha Muhamed-Kheir
Published: December 2019

Strains of
Neisseria meningitidis
of serogroup B isolated in the Czech Republic frequently belong to serotype 22. We analyzed the genetic relationships among strains of this serotype by using the multilocus enzyme electrophoresis technique and the polymorphism of the
pilA
gene. Our results indicate that these strains correspond to a highly heterogeneous population rather than to the expansion of a single clone.
Detection and Characterization of Shiga Toxigenic
Escherichia coli
by Using Multiplex PCR Assays for
stx
1
,
stx
2
,
eaeA
, Enterohemorrhagic
E. coli hlyA
,
rfb
O111
, and
rfb
O157
Authors: Paton Adrienne W. , Paton James C.
Published: December 2019

Shiga toxigenic
Escherichia coli
(STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of
stx
1
,
stx
2
(including variants of
stx
2
),
eaeA
, and enterohemorrhagic
E. coli hlyA
, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the
rfb
(O-antigen-encoding) regions of
E. coli
serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively. The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic
E. coli
strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.
Corynebacterium riegeliisp. nov., an Unusual Species Isolated from Female Patients with Urinary Tract Infections
Authors: Funke Guido , Lawson Paul A. , Collins Matthew D.
Published: December 2019

Four strains of an unknown coryneform bacterium were isolated in pure culture from females with urinary tract infections. Strong urease activity and the ability to slowly ferment maltose but not glucose were the most significant phenotypic features of this catalase-positive, nonmotile, nonlipophilic, rod-shaped bacterium which served to distinguish it from all other presently defined coryneform bacteria. Chemotaxonomic investigations demonstrated that the unknown bacterium belonged to the genusCorynebacterium. Comparative 16S rRNA gene sequence analysis revealed that the isolates were genealogically identical and represented a new subline within the genusCorynebacterium, for which the designationCorynebacterium riegeliisp. nov. is proposed. The type strain ofCorynebacterium riegeliiis CCUG 38180 (DSM 44326, CIP 105310).
Controlled Clinical Laboratory Comparison of Two Supplemented Aerobic and Anaerobic Media Used in Automated Blood Culture Systems To Detect Bloodstream Infections
Authors: Ziegler R. , Johnscher I. , Martus P. , Lenhardt D. , Just H.-M.
Published: December 2019

A 20-ml blood sample was collected from adult patients with suspected bloodstream infections and distributed equally into the four volume-controlled bottles of a blood culture set consisting of aerobic and anaerobic BACTEC Plus/F bottles and aerobic and anaerobic BacT/Alert FAN bottles. All bottles were incubated in their respective instruments for a standard 5-day protocol or until the instruments signalled positivity. Samples in all bottles with negative results by these instruments were terminally subcultured. A total of 8,390 blood culture sets were obtained during the study period, of which 4,402 (52.5%) met the study criteria. Of these, 946 (21.5%) were positive either by instrument signal or by additional terminal subculture of all negative bottles and yielded growth of microorganisms. Five hundred eighty-nine (13.4%) blood culture sets were considered to have recovered 663 clinically significant organisms. When both the BACTEC and the BacT/Alert systems were used, 465 positive sets were detected; BACTEC alone detected 52 positive sets and BacT/Alert alone detected 72 (
P
= 0.09). No differences were found between the two systems in microbial recovery rate from blood cultures obtained from patients on antibiotic therapy. Significantly more members of the family
Enterobacteriaceae
(
P
< 0.01) were detected from patients without antimicrobial therapy by BacT/Alert than by BACTEC. The false-negative rates were 0.20% for BACTEC and 0.32% for BacT/Alert. A significantly higher false-positive rate was found for BACTEC (
P
< 0.0001). Both systems were comparable for the time to detection of microorganisms. However, gram-positive bacteria were detected faster by BACTEC and
Enterobacteriaceae
were detected faster on average by BacT/Alert. We concluded that both systems are comparable in their abilities to recover aerobic and anaerobic organisms from blood cultures and a terminal subculture might not be necessary for either of the two systems. The increased positivity rate when using an anaerobic bottle in a two-bottle blood culture set is due to the additional blood volume rather than to the use of an anaerobic medium.
Rapid Detection of
Mycobacterium paratuberculosis
in Clinical Samples from Ruminants and in Spiked Environmental Samples by Modified BACTEC 12B Radiometric Culture and Direct Confirmation by IS
900
PCR
Authors: Whittington R. J. , Marsh I. , Turner M. J. , McAllister S. , Choy E. , Eamens G. J. , Marshall D. J. , Ottaway S.
Published: December 2019

The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of
Mycobacterium paratuberculosis
in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of
Mycobacterium paratuberculosis
by IS
900
PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate
M. paratuberculosis
from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne’s disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with
M. paratuberculosis
. The results suggested that radiometric culture was more sensitive than histopathology in detecting
M. paratuberculosis
infection in sheep and goats and more sensitive than culture on Herrold’s egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of ≥10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of ≥10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.
Clinical Comparison of an Enhanced-Sensitivity Branched-DNA Assay and Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Nolte Frederick S. , Boysza Jodi , Thurmond Cathy , Clark W. Scott , Lennox Jeffrey L.
Published: December 2019

The performance characteristics of an enhanced-sensitivity branched-DNA assay (bDNA) (Quantiplex HIV-1 version 2.0; Chiron Corp., Emeryville, Calif.) and a reverse transcription (RT)-PCR assay (AMPLICOR HIV-1 Monitor; Roche Diagnostic Systems, Inc., Branchburg, N.J.) were compared in a molecular diagnostic laboratory. Samples used in this evaluation included linearity and reproducibility panels made by dilution of a human immunodeficiency virus type 1 (HIV-1) stock culture of known virus particle count in HIV-1-negative plasma, a subtype panel consisting of HIV-1 subtypes A through F at a standardized level, and 64 baseline plasma specimens from HIV-1-infected individuals. Plots of log
10
HIV RNA copies per milliliter versus log
10
nominal virus particles per milliliter demonstrated that both assays were linear over the stated dynamic ranges (bDNA,
r
= 0.98; RT-PCR,
r
= 0.99), but comparison of the slopes of the regression lines (bDNA,
m
= 0.96; RT-PCR,
m
= 0.83) suggested that RT-PCR had greater proportional systematic error. The between-run coefficients of variation for bDNA and RT-PCR were 24.3 and 34.3%, respectively, for a sample containing 1,650 nominal virus particles/ml and 44.0 and 42.7%, respectively, for a sample containing 165 nominal virus particles/ml. Subtypes B, C, and D were quantitated with similar efficiencies by bDNA and RT-PCR; however, RT-PCR was less efficient in quantitating subtypes A, E, and F. One non-B subtype was recognized in our clinical specimens based on the ratio of values obtained with the two methods. HIV-1 RNA was quantitated in 53 (83%) baseline plasma specimens by bDNA and in 55 (86%) specimens by RT-PCR. RT-PCR values were consistently greater than bDNA values, with population means of 142,419 and 67,580 copies/ml, respectively (
P
< 0.01). The results were highly correlated (
r
= 0.91), but the agreement was poor (mean difference in log
10
copies per milliliter ± 2 standard deviations, 0.45 ± 0.61) for the 50 clinical specimens that gave discrete values with both methods.
Genomic Comparisons and Shiga Toxin Production among
Escherichia coli
O157:H7 Isolates from a Day Care Center Outbreak and Sporadic Cases in Southeastern Wisconsin
Authors: Gouveia S. , Proctor M. E. , Lee M.-S. , Luchansky J. B. , Kaspar C. W.
Published: December 2019

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of
Escherichia coli
O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE using
Xba
I identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine
Xba
I REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential
E. coli
O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (
stx
1
and
stx
2
, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses of
E. coli
O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by
E. coli
O157:H7 strains endemic to eastern Wisconsin.