bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonisotopic Detection and Typing of Human Papillomavirus DNA in Genital Samples by the Line Blot Assay
Authors: Coutlée François , Gravitt Patti , Richardson Harriet , Hankins Catherine , Franco Eduardo , Lapointe Normand , Voyer Hélène ,   The Canadian Women’s HIV Study Group
Published: December 2019

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for β-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43.5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of β-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.
Evaluation of a Nested Reverse Transcription-PCR Assay Based on the Nucleoprotein Gene for Diagnosis of Spontaneous and Experimental Bovine Respiratory Syncytial Virus Infections
Authors: Valarcher Jean-François , Bourhy Hervé , Gelfi Jacqueline , Schelcher François
Published: December 2019

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.
Evidence of High-Frequency Genomic Reassortment of Group A Rotavirus Strains in Bangladesh: Emergence of Type G9 in 1995
Authors: Unicomb Leanne E. , Podder Goutam , Gentsch Jon R. , Woods Patricia A. , Hasan K. Zahid , Faruque A. S. G. , Albert M. John , Glass Roger I.
Published: December 2019

We characterized 1,534 rotavirus (RV) strains collected in Bangladesh from 1992 to 1997 to assess temporal changes in G type and to study the most common G and P types using reverse transcription-PCR, oligonucleotide probe hybridization, and monoclonal antibody-based enzyme immunoassay. Results from this study combined with our previous findings from 1987 to 1991 (F. Bingnan et al., J. Clin. Microbiol. 29:862–868, 1991, and L. E. Unicomb et al., Arch. Virol. 132:201–208, 1993) (
n
= 2,515 fecal specimens) demonstrated that the distribution of the four major G types varied from year to year, types G1 to G4 constituted 51% of all strains tested (
n
= 1,364), and type G4 was the most prevalent type (22%), followed by type G2 (17%). Of 351 strains tested for both G and P types, three globally common types, type P[8], G1, type P[4], G2, and type P[8], G4, comprised 45% (
n
= 159) of the strains, although eight other strains were circulating during the study period. Mixed G and/or P types were found in 23% (
n
= 79) of the samples tested. Type G9 RVs that were genotype P[6] and P[8] with both long and short electrophoretic patterns emerged in 1995. The finding of five different genotypes among G9 strains, of which three were frequently detected, suggests that they may have an unusual propensity for reassortment that exceeds that found among the common G types. We also detected antigenic changes in serotypes G2 and G4 over time, as indicated by the loss of reactivity with standard typing monoclonal antibodies. Our data suggest that a vaccine must provide protection against type G9 RVs as well as against the four major G types because G9 strains constituted 16% (
n
= 56) of the typeable RV strains and have predominated since 1996.
Detection of an Archaic Clone of
Staphylococcus aureus
with Low-Level Resistance to Methicillin in a Pediatric Hospital in Portugal and in International Samples: Relics of a Formerly Widely Disseminated Strain?
Authors: Sá-Leão Raquel , Santos Sanches Ilda , Dias Dora , Peres Isabel , Barros Rosa M. , de Lencastre Hermínia
Published: December 2019

Close to half of the 878 methicillin-resistant
Staphylococcus aureus
(MRSA) strains recovered between 1992 and 1997 from the pediatric hospital in Lisbon were bacteria in which antibiotic resistance was limited to β-lactam antibiotics. The other half were multidrug resistant. The coexistence of MRSA with such unequal antibiotic resistance profiles prompted us to use molecular typing techniques for the characterization of the MRSA strains. Fifty-three strains chosen randomly were typed by a combination of genotypic methods. Over 90% of the MRSA strains belonged to two clones: the most frequent one, designated the “pediatric clone,” was reminiscent of historically “early” MRSA: most isolates of this clone were only resistant to β-lactam antimicrobials and remained susceptible to macrolides, quinolones, clindamycin, spectinomycin, and tetracycline. They showed heterogeneous and low-level resistance to methicillin (MIC, 1.5 to 6 μg/ml), carried the
Cla
I-
mecA
polymorph II, were free of the transposon Tn
554
, and showed macrorestriction pattern D (clonal type II::NH::D). The second major clone was the internationally spread and multiresistant “Iberian” MRSA with homogeneous and high-level resistance to methicillin (MIC, >200 μg/ml) and clonal type I::E::A. Surprisingly, the multidrug-resistant and highly epidemic Iberian MRSA did not replace the much less resistant pediatric clone during the 6 years of surveillance. The pediatric clone was also identified among contemporary MRSA isolates from Poland, Argentina, The United States, and Colombia, and the overwhelming majority of these were also associated with pediatric settings. We propose that the pediatric MRSA strain represents a formerly widely spread archaic clone which survived in some epidemiological settings with relatively limited antimicrobial pressure.
Use of Pulsed-Field Gel Electrophoresis for Molecular Epidemiologic and Population Genetic Studies of
Mycobacterium tuberculosis
Authors: Singh Samir P. , Salamon Hugh , Lahti Carol J. , Farid-Moyer Mehran , Small Peter M.
Published: December 2019

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology of
Mycobacterium tuberculosis
. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of
M. tuberculosis
. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS
6110
RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of
M. tuberculosis
were genotyped with four different PFGE enzymes (
Ase
I,
Dra
I,
Spe
I, and
Xba
I), as well as by RFLP analysis with IS
6110
. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS
6110
typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS
6110
and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS
6110
RFLP genotyping is dependent upon the number of IS
6110
elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that
M. tuberculosis
is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of
M. tuberculosis
.
Distribution of aNocardia brasiliensisCatalase Gene Fragment in Members of the GeneraNocardia,Gordona, andRhodococcus
Authors: Vera-Cabrera Lucio , Johnson Wendy M. , Welsh Oliverio , Resendiz-Uresti Francisco L. , Salinas-Carmona Mario C.
Published: December 2019

An immunodominant protein fromNocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase fromStreptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3′-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in severalN. brasiliensisstrains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the generaNocardia,Rhodococcus,Gordona, andStreptomyces. All of theN. brasiliensisstrains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in theN. brasiliensisstrains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.
Evaluation of the ImmunoCardSTAT! Rotavirus Assay for Detection of Group A Rotavirus in Fecal Specimens
Authors: Dennehy Penelope H. , Hartin Michele , Nelson Sara M. , Reising Shirley F.
Published: December 2019

Rapid detection of group A rotavirus was performed by using ImmunoCardStat! Rotavirus (ICS-RV) (which uses immunogold-based, horizontal-flow membrane technology), two commercial enzyme immunoassays (Premier Rotaclone and TestPack Rotavirus), and electron microscopy. A total of 249 stool specimens collected from children with gastroenteritis between February and April 1997 were tested. After resolution of 19 of the 22 discordant results by reverse transcription-PCR for group A rotavirus, ICS-RV detected 125 positives while Rotaclone and TestPack detected 127 and 129 positives, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value were 94.0, 100, 100, and 93.4% for ICS-RV; 95.5, 100, 100, and 95.0% for Rotaclone; and 97.0, 96.5, 97.0, and 96.5% for TestPack. ICS-RV was sensitive and specific and was relatively simple to perform and interpret.
Activity of Gatifloxacin against
Haemophilus influenzae
and
Moraxella catarrhalis
, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for
H. influenzae
Authors: Jones Ronald N. , Biedenbach Douglas J. , Erwin Meredith E. , Beach Mondell L. , Pfaller Michael A. ,   The Quality Control Study Group
Published: December 2019

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against
Haemophilus influenzae. Moraxella catarrhalis
(600 strains) and
H. influenzae
(1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC
90
], ≤0.03 μg/ml; 100.0% of strains inhibited at ≤2 μg/ml) was the most active agent tested against
H. influenzae
and was similar to four comparison fluoroquinolones (MICs, ≤0.03 to 2 μg/ml) against
M. catarrhalis
. A subset of 300 recent clinical isolates of
H. influenzae
were tested by using media (Haemophilus Test Medium agar and broth) and procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and with the E-test (AB BIODISK, Solna, Sweden). Gatifloxacin (MIC
50
, 0.008 μg/ml) was slightly more active than levofloxacin, and E-test results were generally elevated by 0.5 log
2
dilution step compared to reference MICs. The gatifloxacin 5-μg disk test produced zone diameters that were routinely above 30 mm for
H. influenzae
strains, corresponding to gatifloxacin MICs of 0.008 or 0.016 μg/ml. The gatifloxacin susceptibility breakpoint proposed for nonfastidious species (≤2 μg/ml; ≥18 mm) was also suggested for
H. influenzae
testing. No interpretive errors were observed. Quality control guidelines for
H. influenzae
ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 μg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-
Haemophilus
fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).
Immunochromatography Test for Rapid Diagnosis of Adenovirus Respiratory Tract Infections: Comparison with Virus Isolation in Tissue Culture
Authors: Tsutsumi Hiroyuki , Ouchi Kazunobu , Ohsaki Masaya , Yamanaka Tatsuru , Kuniya Yoshinori , Takeuchi Yoshinao , Nakai Chiaki , Meguro Hidenori , Chiba Shunzo
Published: December 2019

The sensitivity and the specificity of a new commercial rapid 10-min adenovirus antigen immunochromatography (IC) test were determined by comparison with the sensitivity and specificity of virus isolation. Of 169 pharyngeal swabs from children with suspected adenovirus respiratory tract infections, 95 (56%) were culture positive for adenovirus. The IC test was sensitive (detecting 69 of these 95 infections [72.6%]) and completely specific (identifying 74 of 74 specimens [100%]) when it was compared with cell culture. The test detected adenovirus serotypes 1, 2, 3, 5, and 7 with almost equal sensitivities. This test is not only rapid and easy to perform but also sensitive and specific for adenovirus respiratory tract infections. The test is sufficiently rapid to be used at the bedside or in an outpatient clinic, with the result being available during a patient’s first examination.
Typing of Bovine Viral Diarrhea Viruses Directly from Blood of Persistently Infected Cattle by Multiplex PCR
Authors: Gilbert S. A. , Burton K. M. , Prins S. E. , Deregt D.
Published: December 2019

A nested multiplex PCR was developed for genotyping of bovine viral diarrhea viruses (BVDVs). The assay could detect as little as 3 50% tissue culture infective doses of BVDV per ml and typed 42 out of 42 cell culture isolates. BVDV was also successfully typed, with or without RNA extraction, from all 27 whole-blood samples examined from 22 carriers or probable carriers and 5 experimentally infected cattle.
Oxacillin Susceptibility Testing of Staphylococci Directly from Bactec Plus Blood Cultures by the BBL Crystal MRSA ID System
Authors: Kubina M. , Jaulhac B. , Delabranche X. , Lindenmann C. , Piemont Y. , Monteil H.
Published: December 2019

The BBL Crystal MRSA ID test (Becton Dickinson) was applied directly to blood culture vials containing clusters of gram-positive cocci. The sensitivity and specificity of the test were 84 and 100% and 54 and 100% for vials containing
Staphylococcus aureus
and coagulase-negative staphylococci, respectively. This test is a reliable method for direct detection of methicillin resistance in positive blood culture vials when
S. aureus
is identified in parallel by rapid identification procedures.
Simple Strategy for Direct Identification of Medically Important Yeast Species from Positive Blood Culture Vials
Authors: Sheppard Donald C. , René Pierre , Harris Anthony D. , Miller Mark A. , Laverdière Michel , deSouza Edwige , Robson Hugh G.
Published: December 2019

We compared direct inoculation of the Auxacolor yeast identification system from positive blood culture vials to standard identification with the API 20C AUX (API 20C), using 44 prospectively collected clinical specimens and 25 seeded blood culture vials. Direct inoculation of the Auxacolor system was accurate and more rapid than standard identification with the API 20C.
Enhanced Amplified Mycobacterium Tuberculosis Direct Test for Detection of
Mycobacterium tuberculosis
Complex in Positive BACTEC 12B Broth Cultures of Respiratory Specimens
Authors: Bergmann John S. , Woods Gail L.
Published: December 2019

The reliability of the Gen-Probe enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD) for identification of
Mycobacterium tuberculosis
complex (MTBC) in BACTEC 12B broth cultures of respiratory specimens was evaluated by testing aliquots from 268 bottles with a growth index of ≥50. MTD results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen-Probe, Inc.) testing sediment from broth samples or colonies on a solid medium. For the first 134 cultures, from which 68 mycobacterial isolates (including 27 MTBC isolates) were recovered, both fresh and frozen aliquots were tested. MTD results for the frozen aliquots agreed with the identification by usual protocol in all cases, whereas there was one false-negative MTD result with fresh aliquots. For the remaining 134 cultures, only frozen aliquots were tested. Of the total 268 broth cultures (from 210 patients) evaluated, 137 (51.1%) grew mycobacteria, including 60 MTBC isolates. All 60 isolates were MTD positive, as was one additional culture that grew
Mycobacterium gordonae
. The latter culture was from a patient who was diagnosed with tuberculosis a few months earlier and was on therapy; therefore, the MTD result was considered a true positive. Sensitivity, specificity, and positive and negative predictive values of MTD were 100%. The mean times from specimen receipt to identification of MTBC were 15 (±1) days (range, 4 to 27 days) for BACTEC plus MTD and 19 (±1) days (range, 6 to 36 days) for the usual protocol (
P
< 0.001). These data indicate that the MTD is a rapid, reliable method for identification of MTBC in fresh or frozen aliquots of broth from positive BACTEC 12B cultures of respiratory specimens.
Molecular and Epidemiological Characterization of Vaginal
Saccharomyces cerevisiae
Isolates
Authors: Posteraro Brunella , Sanguinetti Maurizio , D’Amore Giuseppina , Masucci Luca , Morace Giulia , Fadda Giovanni
Published: December 2019

Although vaginitis caused by
Saccharomyces cerevisiae
is extremely rare, in recent years we have experienced an increasing frequency of
S. cerevisiae
isolation from the vaginas of fertile-age women. In order to investigate the epidemiology of these vaginal infections, a total of 40 isolates of
S. cerevisiae
derived from symptomatic and asymptomatic women were characterized by two DNA typing approaches, named ribosomal DNA (rDNA) hybridization and Ty917 hybridization, based on the Southern blotting technique. After transfer, the polymorphic DNA restriction fragments were hybridized with the entire repeat of
S. cerevisiae
rDNA for one method and with the entire sequence of the Ty917 retrotransposon for the other. After elaboration with computer-assisted analysis, the results of each method showed that Ty917 hybridization is endowed with a discriminatory power higher than that of rDNA hybridization. With the Ty917 hybridization method, all of the
S. cerevisiae
isolates tested appeared very heterogeneous, with the exception of those collected from individual patients with recurrent vaginitis. This allowed us to exclude a possible common source of infection while the high relatedness among
S. cerevisiae
sequential isolates from recurrent-vaginitis patients could suggest a pattern of relapse rather than frequent reinfection.
Detection of
Helicobacter pylori
DNA in Fecal Samples from Infected Individuals
Authors: Gramley William A. , Asghar Ali , Frierson Henry F. , Powell Steven M.
Published: December 2019

Stool, gastric biopsy, and serum samples were collected from 22 subjects. DNA from stool was extracted, amplified, and hybridized with primers specific for the 16S rRNA gene of
Helicobacter pylori
. DNA from gastric biopsy specimens was analyzed similarly for comparison. Universal primers were used to confirm successful extraction of DNA from samples. Histologic, serologic, and DNA analyses were scored in a blinded fashion. Universal primer amplification verified successful DNA extraction from all stool and gastric tissue specimens. The gastric tissue DNA assay was positive for
H. pylori
in 11 of the 22 subjects, correlating completely with histologic and serologic results. Stool DNA was positive for
H. pylori
by our molecular assay in 8 of these 11
H. pylori
-positive subjects. All subjects who were negative by histologic, serologic, and gastric tissue DNA analyses were also negative by stool DNA analysis. Compared to histology, serology, and gastric tissue DNA analyses, the sensitivity of our stool DNA assay was 73%, with a specificity of 100%.
Detection of Human Papillomavirus Types 6 and 11 in Pubic and Perianal Hair from Patients with Genital Warts
Authors: Boxman Ingeborg L. A. , Hogewoning Arjan , Mulder Linda H. C. , Bavinck Jan Nico Bouwes , ter Schegget Jan
Published: December 2019

Genital human papillomavirus (HPV) types 6 and 11 are of clinical importance due to their role in the development of anogenital warts. A pilot study was performed to investigate whether DNAs from HPV types 6 and 11 are present in hairs plucked from the pubic and perianal regions and eyebrows of patients with genital warts at present and patients with a recent history of genital warts. Genital HPV DNA was detected in 9 of 25 (36%) pubic hair samples and in 11 of 22 (50%) perianal hair samples by the CPI/CPIIg PCR. After sequencing of 17 of 20 samples, HPV type 6 or 11 was detected in 6 of 25 (24%) hair samples from the pubis and 8 of 22 (36%) hair samples from the perianal region. These types were not detected in plucked eyebrow hairs. In contrast, the HPV types associated with epidermodysplasia verruciformis were detected in similar proportions (62%) in both samples of pubic and eyebrow hairs. Moreover, HPV type 6 and 11 DNAs were detected in pubic hairs plucked from two patients who had been successfully treated and who did not show any lesion at the time of hair collection; this finding is an argument that HPV DNA may persist in this region. The presence of genital HPV types in plucked pubic and perianal hair suggests that there is an endogenous reservoir for HPV which may play a role in the recurrences of genital warts.
Detection of Equine Antibodies toBabesia caballiby RecombinantB. caballiRhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay
Authors: Kappmeyer Lowell S. , Perryman Lance E. , Hines Stephen A. , Baszler Timothy V. , Katz Jonathan B. , Hennager Steven G. , Knowles Donald P.
Published: December 2019

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific forBabesia caballi. The assay used recombinantB. caballirhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDaB. caballiantigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies toB. caballiby a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive withB. caballiRAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive withB. caballiwhen they were tested by IFA. These data indicate that following infection withB. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballiantibodies.
Distinct Variants of
Helicobacter pylori cagA
Are Associated with
vacA
Subtypes
Authors: van Doorn Leen-Jan , Figueiredo Céu , Sanna Ricardo , Blaser Martin J. , Quint Wim G. V.
Published: December 2019

The diversity of the cytotoxin-associated gene (
cagA
) of
Helicobacter pylori
was analyzed in 45 isolates obtained from nine countries. We examined variation in the 5′ end of the
cagA
open reading frame as determined by PCR and sequencing. Phylogenetic analysis revealed the existence of at least two distinct types of
cagA
. One variant (
cagA1
) was found exclusively in strains from Europe, the United States, and Australia, whereas a novel variant (
cagA2
) was found in strains from East Asia. The greatest diversity between
cagA1
and
cagA2
was found in the first 20 amino acids of the
cagA
open reading frame, where several consistent insertions or deletions were observed. Additional
cagA
sequence variants that could be classified as separate subtypes were found in two of three Peruvian and in five of seven U.S. strains tested. The calculated isoelectric point of the first 154 amino acids of the
cagA1
variants (7.52 ± 1.54) was significantly higher than that of the first 154 amino acids of the
cagA2
variants (5.61 ± 0.94;
P
< 0.001). Most
cagA2
strains contained
vacA
subtype s1c (
P
< 0.001), and in
vacA
m1 strains
cagA1
was more frequently observed than
cagA2
. These results show the epidemiological relationship between
cagA
and
vacA
at the subtype level and indicate the existence of distinct
H. pylori
lineages that are not uniformly distributed over the globe.
Dissemination of a Chloramphenicol- and Tetracycline-Resistant but Penicillin-Susceptible Invasive Clone of Serotype 5
Streptococcus pneumoniae
in Colombia
Authors: Tamayo Mónica , Sá-Leão Raquel , Sanches Ilda Santos , Castañeda Elizabeth , de Lencastre Hermínia
Published: December 2019

A national surveillance conducted in Colombia between 1994 and 1996 identified serotype 5
Streptococcus pneumoniae
as the second most frequent cause of invasive disease in children younger than 5 years of age. All 43 serotype 5 isolates collected during this period were shown to be susceptible to penicillin, erythromycin, cefotaxime, and vancomycin, but most (38 of 43, or 88%) were highly resistant to chloramphenicol. In order to clarify a possible genetic relatedness among these isolates, additional microbiological and molecular characterizations were performed. Most (40 of 43, or 93%) of the isolates were found to be resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) patterns of chromosomal DNAs revealed that all the 43 isolates were closely related and that 38 of the 43 isolates were representatives of a “Colombian clone” of
S. pneumoniae
isolates which were recovered throughout the 3-year surveillance period from patients in 13 hospitals located in five Colombian cities. Isolates belonging to this Colombian clone were resistant to chloramphenicol and tetracycline, hybridized with the
cat
and
tetM
DNA probes in the same 340-kb
Sma
I fragment, and had identical PFGE patterns after both
Sma
I and
Apa
I digestions.
Analysis of
vacA
,
cagA
, and IS
605
Genotypes and Those Determined by PCR Amplification of DNA between Repetitive Sequences of
Helicobacter pylori
Strains Isolated from Patients with Nonulcer Dyspepsia or Mucosa-Associated Lymphoid Tissue Lymphoma
Authors: van Doorn Nathalie E. M. , Namavar Ferry , van Doorn Leen-Jan , Durrani Zarmina , Kuipers Ernst J. , Vandenbroucke-Grauls Christina M. J. E.
Published: December 2019

The
vacA
s and m genotypes and the presence of
cagA
and IS
605
were determined in
Helicobacter pylori
strains from patients with mono- and multiple infections. Surprisingly, these genetic markers were not associated with nonulcer dyspepsia or mucosa-associated lymphoid tissue lymphoma. The presence of
cagA
correlated with the presence of the
vacA
s1 allele (
P
< 0.05), whereas the presence of IS
605
was associated with the presence of the s2 allele (
P
< 0.05).
An Untypeable
Shigella flexneri
Strain Associated with an Outbreak in California
Authors: Trevejo Rosalie T. , Abbott Sharon L. , Wolfe Mitchell I. , Meshulam Jerry , Yong David , Flores George R.
Published: December 2019

Eleven
Shigella flexneri
(group B) isolates were recovered from epidemiologically linked patrons and food handlers from a restaurant-associated outbreak of shigellosis. Six isolates available for pulsed-field gel electrophoresis were identical. All strains agglutinated in group B and subgroup factor 6 sera but not in group 1 through group 6 sera.
Distribution of Human Rotavirus G Types Circulating in Paris, France, during the 1997–1998 Epidemic: High Prevalence of Type G4
Authors: Gault Elyanne , Chikhi-Brachet Roxane , Delon Sandrine , Schnepf Nathalie , Albiges Laurence , Grimprel Emmanuel , Girardet Jean-Philippe , Begue Pierre , Garbarg-Chenon Antoine
Published: December 2019

Group A human rotavirus G genotypes were determined by means of reverse transcription-PCR in 170 stool specimens from children with acute diarrhea admitted to a Paris children’s hospital during a 1-year survey (1997 to 1998). The isolates all belonged to types G1 to G4, with type G4 predominating (60%).
PCR and Blood Culture for Detection of
Escherichia coli
Bacteremia in Rats
Authors: Heininger Alexandra , Binder Marlies , Schmidt Sibylle , Unertl Klaus , Botzenhart Konrad , Döring Gerd
Published: December 2019

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 10
6
CFU of
Escherichia coli
. Blood was then taken at several time points for detection of
E. coli
by BC and by PCR with
E. coli
-specific primers derived from the
uidA
gene, encoding β-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after
E. coli
challenge. Without this chemotherapy, the
E. coli
detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (
P
, >0.05). Chemotherapy decreased the
E. coli
detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (
P
, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.
Invasion and Intracellular Development of the Human Granulocytic Ehrlichiosis Agent in Tick Cell Culture
Authors: Munderloh Ulrike G. , Jauron Steven D. , Fingerle Volker , Leitritz Lorenz , Hayes S. Fred , Hautman Joan M. , Nelson Curtis M. , Huberty Brent W. , Kurtti Timothy J. , Ahlstrand Gilbert G. , Greig Barbara , Mellencamp Martha A. , Goodman Jesse L.
Published: December 2019

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.
Performance of the Affymetrix GeneChip HIV PRT 440 Platform for Antiretroviral Drug Resistance Genotyping of Human Immunodeficiency Virus Type 1 Clades and Viral Isolates with Length Polymorphisms
Authors: Vahey Maryanne , Nau Martin E. , Barrick Sandra , Cooley John D. , Sawyer Robert , Sleeker Alex A. , Vickerman Peter , Bloor Stuart , Larder Brendan , Michael Nelson L. , Wegner Scott A.
Published: December 2019

The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1)
pol
cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant
pol
gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of
pol
genotypes in future assay versions.
Detection of Phylogenetically Diverse Human Immunodeficiency Virus Type 1 Groups M and O from Plasma by Using Highly Sensitive and Specific Generic Primers
Authors: Yang Chunfu , Pieniazek Danuta , Owen Sherry M. , Fridlund Carol , Nkengasong John , Mastro Timothy D. , Rayfield Mark A. , Downing Robert , Biryawaho Benon , Tanuri Amilcar , Zekeng Leopold , van der Groen Guido , Gao Feng , Lal Renu B.
Published: December 2019

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various
env
subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the “window period.” Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.
Coinfection with Multiple Tick-Borne Pathogens in a Walker Hound Kennel in North Carolina
Authors: Kordick S. K. , Breitschwerdt E. B. , Hegarty B. C. , Southwick K. L. , Colitz C. M. , Hancock S. I. , Bradley J. M. , Rumbough R. , Mcpherson J. T. , MacCormack J. N.
Published: December 2019

Both dogs and humans can be coinfected with variousEhrlichia,Bartonella,Rickettsia, andBabesiaspecies. We investigated a kennel of sick Walker Hounds and their owners in southeastern North Carolina for evidence of tick-borne infections and associated risk factors. A high degree of coinfection was documented in the dog population. Of the 27 dogs, 26 were seroreactive to anEhrlichiasp., 16 toBabesia canis, and 25 toBartonella vinsonii, and 22 seroconverted toRickettsia rickettsiiantigens. According to PCR results, 15 dogs were infected withEhrlichia canis, 9 withEhrlichia chaffeensis, 8 withEhrlichia ewingii, 3 withEhrlichia equi, 9 withEhrlichia platys, 20 with aRickettsiaspecies, 16 with aBartonellaspecies, and 7 withB. canis. The detection of DNA from anyEhrlichiaspecies was associated with clinical illness and with concurrentB. canisinfection (by PCR). BothE. canisand an uncharacterizedRickettsiaspecies appeared to result in chronic or recurrent infection. Death in the dog population was associated with living in a dirt lot rather than the concrete kennel. Of 23 people on whom serologic testing was conducted, eight were seroreactive toBartonella henselae, one toE. chaffeensis, and one toR. rickettsiiantigen; however, none had clinical or hematologic abnormalities consistent with illness caused by these organisms. We conclude that kennel dogs with heavy tick exposure can be infected at a high rate with multiple, potentially zoonotic, tick-borne pathogens. In addition, our findings further illustrate the utility of PCR for documenting coinfection with tick-transmitted pathogens.
Evaluation of the Granada Agar Plate for Detection of Vaginal and Rectal Group B Streptococci in Pregnant Women
Authors: Gil E. García , Rodríguez M. C. , Bartolomé R. , Berjano B. , Cabero L. , Andreu A.
Published: December 2019

Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (
n
= 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (
n
= 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.
Single Clonal Origin of a High Proportion of
Legionella pneumophila
Serogroup 1 Isolates from Patients and the Environment in the Area of Paris, France, over a 10-Year Period
Authors: Lawrence Christine , Reyrolle Monique , Dubrou Sylvie , Forey Françoise , Decludt Bénedicte , Goulvestre Claire , Matsiota-Bernard Peggy , Etienne Jerome , Nauciel Charles
Published: December 2019

Arbitrarily primed PCR with three primers and pulsed-field gel electrophoresis were used to characterize a set of 75 clinical
Legionella pneumophila
serogroup 1 isolates, with no apparent epidemiological link, obtained from 24 hospitals in Paris, France, from 1987 to 1997. Unexpectedly, 25 clinical isolates from 15 hospitals had an identical profile (termed type A) by both methods. The same profile was subsequently found in 16 of 64 randomly selected environmental
L. pneumophila
serogroup 1 isolates from 15 different sites in the Paris area. There was no evidence of geographic clustering or a peak incidence of type A isolation. Type A has not been found in France outside the Paris area, suggesting that a particular type of
L. pneumophila
serogroup 1 is specifically present in the Paris water distribution network.
Novel Immunoblot Assay Using Four Recombinant Antigens for Diagnosis of Epstein-Barr Virus Primary Infection and Reactivation
Authors: Buisson M. , Fleurent B. , Mak M. , Morand P. , Chan L. , Ng A. , Guan M. , Chin D. , Seigneurin J. M.
Published: December 2019

A new immunoblot assay, composed of four Epstein-Barr virus (EBV)-encoded recombinant proteins (virus capsid antigen [VCA] p23, early antigen [EA] p138, EA p54, and EBNA-1 p72), was compared with an immunofluorescence assay on a total of 291 sera. The test was accurate in 94.5% of cases of primary EBV infection, while an immunoglobulin G anti-VCA p23 band with strong intensity correlated with reactivation.
A Multidrug-Resistant Tuberculosis Microepidemic Caused by Genetically Closely Related
Mycobacterium tuberculosis
Strains
Authors: Kubín M. , Havelková M. , Hynčicová I. , Švecová Z. , Kaustová J. , Kremer K. , van Soolingen D.
Published: December 2019

IS
6110
DNA fingerprinting was used to characterize an outbreak of multidrug-resistant tuberculosis in 21 individuals (17 males and 4 females) living in or roaming among four distantly separated areas in the Czech Republic. The restriction fragment length polymorphism (RFLP) analysis separated the collected
Mycobacterium tuberculosis
strains into group A, including 14 patients with six IS
6110
copies, and group B, with 7 patients displaying highly similar RFLP patterns but with two additional IS
6110
bands. A switch from pattern A to pattern B was observed in one patient, and the subsequent detection of subclone B in seven more individuals has been explained by the instability of DNA genotypes caused by transposition of IS
6110
elements.
Typing of Clinical Herpes Simplex Virus Type 1 and Type 2 Isolates with Monoclonal Antibodies
Authors: Liljeqvist Jan-Åke , Svennerholm Bo , Bergström Tomas
Published: December 2019

The purpose of this study was to evaluate the performance of a herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1 monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2 MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively, using an enzyme immunoassay. The anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity, indicating that these MAbs are suitable for typing of clinical HSV isolates.
Sequencing of the Ribosomal Intergenic Spacer Region for Strain Identification of
Porphyromonas gingivalis
Authors: Rumpf Robert W. , Griffen Ann L. , Wen Bo-Gui , Leys Eugene J.
Published: December 2019

The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of
Porphyromonas gingivalis
were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a
P. gingivalis
ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison.