bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Rapid Immunochromatographic Test for Diagnosis of Dengue Virus Infection
Authors: Vaughn David W. , Nisalak Ananda , Kalayanarooj Siripen , Solomon Tom , Dung Nguyen Minh , Cuzzubbo Andrea , Devine Peter L.
Published: December 2019

A rapid (<7-min) immunochromatographic test for immunoglobulin M (IgM) and IgG antibodies to dengue viruses was evaluated by using hospital admission and discharge sera from 124 patients. The reference laboratory diagnosis was based on the results of virus isolation, hemagglutination-inhibition assay (HAI), and enzyme immunoassay (EIA). By the standard assays, patients experienced primary dengue virus infection (
n
= 30), secondary dengue virus infection (
n
= 48), Japanese encephalitis (JE) virus infection (
n
= 20), or no flavivirus infection (
n
= 26). The rapid test demonstrated 100% sensitivity in the diagnosis of dengue virus infection and was able to distinguish between primary and secondary dengue virus infections through the separate determinations of IgM and IgG. For all patients with primary dengue virus infection a positive test for IgM to dengue virus and a negative test for IgG to dengue virus were obtained, whereas for 46 of 48 patients (96%) with secondary dengue virus infection, a positive test for IgG to dengue virus with or without a positive test for IgM to dengue virus was obtained. The remaining two patients with secondary dengue virus infection had positive IgM test results and negative IgG test results. Furthermore, the rapid test was positive for patients confirmed to be infected with different dengue virus serotypes (12 infected with dengue virus serotype 1, 4 infected with dengue virus serotype 2, 3 infected with dengue virus serotype 3, and 2 infected with dengue virus serotype 4). The specificity of the test for nonflavivirus infections was 88% (3 of 26 positive), while for JE virus infections the specificity of the test was only 50% (10 of 20). However, most patients with secondary dengue virus infection were positive for both IgM and IgG antibodies to dengue virus, while no patients with JE virus infection had this profile, so cross-reactivity was only a concern for a small proportion of patients with secondary dengue infections. The rapid test demonstrated a good correlation with the reference EIA and HAI and should be useful for the rapid diagnosis of dengue virus infections.
Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by Using Blood Dried on Filter Paper
Authors: Fiscus Susan A. , Brambilla Donald , Grosso Lynell , Schock Jody , Cronin Michael
Published: December 2019

We compared whole blood dried on filter paper to the standard assay with frozen cell-free plasma for use in the quantitation of the human immunodeficiency virus RNA load in blood. RNA values from filter paper, corrected for the hematocrit, gave results comparable to those of the standard assay in terms of sensitivity and reproducibility.
Inhibition of PCR by Agar from Bacteriological Transport Media
Authors: Gibb Alan Patrick , Wong Sallene
Published: December 2019

We observed inhibition of PCR in throat swabs submitted in routine bacteriological transport media. Experimental studies showed that agar, which was extracted with DNA by DNAzol (Gibco BRL, Gaithersburg, Md.), was the inhibitory agent. No inhibitory effect was observed with a transport medium which did not contain agar.
Pulsed-Field Gel Electrophoresis of Genomic Digests Demonstrates Linkages among Food, Food Handlers, and Patrons in a Food-Borne
Salmonella javiana
Outbreak in Massachusetts
Authors: Lee Roger , Peppe Joseph , George Harvey
Published: December 2019

A total of 66 isolates of
Salmonella javiana
isolated from food, food handlers, and patrons that were epidemiologically linked to an outbreak of gastroenteritis were analyzed by pulsed-field gel electrophoresis. Analysis with restriction endonucleases
Xba
I and
Spe
I supported the epidemiologic association and suggested a pathway of transmission among food, food handlers, and patrons.
Use of a Gastric Juice-Based PCR Assay To Detect
Helicobacter pylori
Infection in Culture-Negative Patients
Authors: Yoshida Haruhiko , Hirota Katsutaro , Shiratori Yasushi , Nihei Takeshi , Amano Shin , Yoshida Akira , Kawamata Osamu , Omata Masao
Published: December 2019

A gastric juice-based PCR assay was compared with culture, microscopy, and a rapid urease test with specimens from 114 subjects. The PCR and conventional tests were positive for 76 and 62% of the subjects, respectively. The prevalence of gastroduodenal disease and seropositivity for anti-
Helicobacter pylori
immunoglobulin G were similarly high among conventional-test-positive and PCR-only-positive subjects compared to all-negative ones. The PCR assay is recommended to confirm the
H. pylori
status of culture-negative peptic-ulcer patients.
Detection of
Helicobacter pylori
in Fecal Samples of Gnotobiotic Mice Infected with
H. pylori
by an Immunomagnetic-Bead Separation Technique
Authors: Osaki Takako , Taguchi Haruhiko , Yamaguchi Hiroyuki , Kamiya Shigeru
Published: December 2019

By an immunomagnetic-bead (IMB) separation technique, isolation of
Helicobacter pylori
from gastrointestinal and fecal samples of gnotobiotic mice infected with the microorganism was tried. The isolation rate of
H. pylori
from stomach samples after IMB separation was not higher than that of direct culture of the samples. After IMB separation of feces,
H. pylori
was detectable by PCR, although
H. pylori
was not culturable.
Amplification of Ehrlichial DNA from Dogs 34 Months after Infection withEhrlichia canis
Authors: Harrus Shimon , Waner Trevor , Aizenberg Itzhak , Foley Janet E. , Poland Amy M. , Bark Hylton
Published: December 2019

In order to determine whether dogs in the subclinical phase of canine monocytic ehrlichiosis (CME) are carriers ofEhrlichia canisand to determine the significance of persistent indirect immunofluorescent anti-E. canisantibody titers during this phase, PCR was performed with blood, bone marrow, and splenic aspirates collected 34 months postinoculation from six clinically healthy beagle dogs experimentally infected withE. canis. At least one of the three samples (spleen, bone marrow, and blood) from four of the six dogs was PCR positive. The spleens of all four of these dogs were PCR positive, and the bone marrow and blood of two of the four dogs were PCR positive. Indirect immunofluorescent-antibody titers increased progressively during the first 5 months postinfection, remained high for an additional period of more than 11 months, and declined thereafter, suggesting that the dogs were recovering from the disease. Five of the dogs remained seropositive 34 months postinfection. The data obtained in this study demonstrate for the first time that clinically healthy dogs in the subclinical phase of CME are carriers of the rickettsia. It was shown that dogs can harborE. canisfor years without developing the chronic clinical disease and that dogs can eliminate the parasite and recover from CME without medical treatment. Our findings suggest that the spleen is the organ most likely to harborE. canisparasites during the subclinical phase and the last organ to accommodate the parasite before elimination. It was concluded that PCR of DNA extracted from splenic aspirates is a reliable method for determining the carrier state of CME.
Development of Specific Immunoglobulins G, M, and A Following Primary
Toxoplasma gondii
Infection in Pregnant Women
Authors: Jenum Pål A. , Stray-Pedersen Babill
Published: December 2019

The development of specific antibodies following primary
Toxoplasma gondii
infection during pregnancy was assessed by six different antibody assays: dye test, Platelia Toxo-IgG, Toxo-Screen DA IgG, Platelia Toxo-IgM, Toxo-ISAGA IgM, and Platelia Toxo-IgA. A total of 126 sera from 27 pregnant women, for whom the time of acquisition of infection could be estimated fairly accurately, were included. All tests showed great individual variation in the peak amounts of antibodies detected. The times elapsed after infection until the peak was reached also varied greatly from individual to individual: the ranges were 2 to 21 weeks for the dye test, 4 to 36 weeks for Platelia Toxo-IgG, 4 to 30 weeks for Toxo-Screen DA IgG, 2 to 18 weeks for Platelia Toxo-IgM, 1 to 6 weeks for Toxo-ISAGA IgM, and 2 to 21 weeks for Platelia Toxo-IgA. In the early phase of the infection the dye test and the specific-IgM tests were the most sensitive. Toxo-Screen DA IgG was more sensitive than Platelia Toxo-IgG in the acute phase, while Platelia Toxo-IgA was clearly the least sensitive assay. Of the sera collected 21 to 52 weeks after infection, all were positive by the dye test, all except one (which was negative by Platelia Toxo-IgG) were positive by the specific-IgG tests, approximately 80% were positive by the IgM tests, and 45% were positive by the IgA test. Due to the great individual variation it seems impossible to estimate when the infection occurred based on results obtained from a single serum, and it may even be difficult to assess when a titer increase in paired sera is detectable unless the first sample is only marginally positive. As a diagnostic criterion a dye test titer of ≥300 IU/ml has a low sensitivity for recent primary infection.
Molecular Epidemiology of Penicillin-Resistant
Streptococcus pneumoniae
Isolates Recovered in Italy from 1993 to 1996
Authors: Marchese Anna , Ramirez Mario , Schito Gian Carlo , Tomasz Alexander
Published: December 2019

Thirty-nine penicillin-resistant
Streptococcus pneumoniae
isolates recovered among the approximately 700 pneumococcal strains collected from 1993 to 1996 in central and northern Italy were analyzed for several characteristics, including serotype, antibiotic susceptibility profile, chromosomal relatedness (by using pulsed-field gel electrophoresis [PFGE]), restriction fragment length polymorphism (RFLP) of the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, and the presence of a variety of antibiotic resistance genes (determined by hybridization with appropriate DNA probes). The MICs of penicillin for most of the isolates (30 of 39) were high, in the range of 1 μg/ml or higher, and these 30 isolates carried additional resistance traits to two or more drugs (erythromycin, chloramphenicol, co-trimoxazole, and tetracycline) and expressed serotypes 9, 19, and 23 and three distinct PFGE patterns. More than half (22 of 30) of the isolates for which MICs were high were identified as representatives of two widespread international epidemic clones of
S. pneumoniae
. The first one of these clones (seven isolates) expressed serotype 23F and possessed all properties characteristic of the widespread Spanish/USA international clone. Seven additional strains with serotype 19 also had the same PFGE pattern, PBP gene, and RFLP polymorphisms, and other properties typical of the serotype 23 Spanish/USA clone, suggesting that these strains were the products of a capsular transformation event (from serotype 23F to serotype 19) in which the Spanish/USA clone was the recipient. The second international clone was represented by eight serotype 9 isolates which were resistant to penicillin and trimethoprim-sulfamethoxazole and had the molecular properties of the French/Spanish epidemic clone. The remaining eight isolates for which penicillin MICs were high appeared to represent a hitherto-undescribed “Italian” clone; they had a novel PFGE type, unique RFLPs for the PBP genes, and resistance to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin, and the penicillin MICs for these isolates were 2 to 4 μg/ml.
Field Validation of Laboratory Tests for Clinical Diagnosis of Sheep-Associated Malignant Catarrhal Fever
Authors: Müller-Doblies U. U. , Li H. , Hauser B. , Adler H. , Ackermann M.
Published: December 2019

Until recently, sheep-associated malignant catarrhal fever (SA-MCF) was diagnosed mainly on the basis of clinical presentation and histopathological changes. Using clinically diagnosed field cases, we have evaluated a seminested PCR and a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) and compared these assays in the diagnosis of SA-MCF in cattle with histopathology as a provisional “gold standard.” Samples from 44 cattle with clinical signs suggestive of SA-MCF were examined by histopathology, PCR, and CI-ELISA. In addition, samples from healthy cattle were evaluated by PCR (
n
= 96) and CI-ELISA (
n
= 75). Based on histopathology, 38 of the 44 clinical cases were classified as SA-MCF positive, 3 were classified as inconclusive, and 3 were classified as SA-MCF negative. The sensitivity of PCR was 95 to 97%, whereas the specificity ranged between 94 and 100%. The CI-ELISA showed a sensitivity of 56 to 87% and a specificity between 91 and 100%. In the field, there is good correlation between the diagnoses of SA-MCF by histopathology, PCR, and CI-ELISA. These data also confirm the close association of ovine herpesvirus 2 with SA-MCF in Switzerland.
Hepatitis C Virus Serotype-Specific Core and NS4 Antibodies in Injecting Drug Users Participating in the Amsterdam Cohort Studies
Authors: Beld Marcel , Penning Maarten , van Putten Marieke , van den Hoek Anneke , Lukashov Vladimir , McMorrow Martin , Goudsmit Jaap
Published: December 2019

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75.5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5′ untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.
Evaluation of a Selective Transport Medium for Gastric Biopsy Specimens To Be Cultured for
Helicobacter pylori
Authors: Siu L. K. , Leung W. K. , Cheng A. F. B. , Sung J. Y. , Ling T. K. W. , Ling J. M. L. , Ng E. K. W. , Lau J. Y. W. , Chung S. C. S.
Published: December 2019

Since the means of culturing
Helicobacter pylori
may not be available in some laboratories, prolonging the survival of this organism during transportation is a major concern in terms of improving detection rates. A selective transport medium was evaluated for the preservation of
H. pylori
from 254 gastric biopsy specimens collected from a rural area in China where culturing is not feasible. Gastric biopsy specimens were inoculated in sterile broth consisting of brain heart infusion (BHI) broth, horse serum, and yeast extract supplemented with vancomycin, amphotericin B, and nalidixic acid (VAN). Of the 254 biopsy specimens, 238 were identified by histology to have
H. pylori
infection. Total rates of recovery of
H. pylori
from the
H. pylori
-positive gastric biopsy specimens stored in the BHI-VAN broth ranged from 76 to 46% after storage of specimens for 5 to 9 days. In conclusion, the selective medium is useful for prolonging the survival of
H. pylori
in gastric biopsy specimens for which immediate culture is not feasible.
Genetic Relationship between Blood and Nonblood Isolates from Bacteremic Patients Determined by Pulsed-Field Gel Electrophoresis
Authors: Matsuda Junichi , Hirakata Yoichi , Iori Fumiaki , Mochida Chikako , Ozaki Yumi , Nakano Michiko , Izumikawa Kohichi , Yamaguchi Toshiyuki , Yoshida Ryoji , Miyazaki Yoshitsugu , Maesaki Shigefumi , Tomono Kazunori , Yamada Yasuaki , Kohno Shigeru , Kamihira Shimeru
Published: December 2019

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis. Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram-positive cocci and 42.1% with
Pseudomonas aeruginosa
bacteremia, indicating that antibiograms of a single nonblood
P. aeruginosa
isolate are not always informative for treatment of bacteremia.
Identification of
Brucella
by Ribosomal-Spacer-Region PCR and Differentiation of
Brucella canis
from Other
Brucella
spp. Pathogenic for Humans by Carbohydrate Profiles
Authors: Fox Karen F. , Fox Alvin , Nagpal Madan , Steinberg Paul , Heroux Karen
Published: December 2019

Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms. The genus
Brucella
consists of a highly conserved group of organisms. Identification of the four species pathogenic in humans (
Brucella melitensis
,
Brucella abortus
,
Brucella suis
, and
Brucella canis
) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species. PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism.
B. abortus
,
B. melitensis
,
B. suis
, and
B. canis
produced identical PCR interspace profiles. However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including
Bartonella
spp. and
Agrobacterium
spp.). Carbohydrate profiles differentiated
B. canis
from the other three
Brucella
species due to the absence of the rare amino sugar quinovosamine in the three other species. PCR of the rRNA interspace region is useful in identification of the genus
Brucella
, while carbohydrate profiling is capable of differentiating
B. canis
from the other
Brucella
species.
Performance of the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay in Detecting
Chlamydia trachomatis
in Endocervical and Urine Specimens from Women and Urethral and Urine Specimens from Men Attending Sexually Transmitted Disease and Family Planning Clinics
Authors: Ferrero Dennis V. , Meyers Holly N. , Schultz Diane E. , Willis Stephen A.
Published: December 2019

The Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) uses transcription-mediated amplification and hybridization protection assay procedures to qualitatively detect
Chlamydia trachomatis
rRNA in urine, endocervical swab, and urethral specimens. The performance of the AMP CT was compared to that of cell culture for endocervical swab and urine specimens from women and urethral and urine specimens from men. Analysis of specimens with discrepant results was performed by a combination of reculture, direct fluorescent-antibody (DFA) staining of specimen sediment, and amplification which targeted a different chlamydial rRNA. A total of 800 urine samples were tested by the AMP CT (607 from women and 193 from men), and 7.1% were positive for
C. trachomatis
, with a sensitivity of 91.2% and a specificity of 99.6% upon discrepant analysis. A total of 926 swab specimens were tested by culture and AMP CT (717 endocervical swab specimens and 209 urethral swab specimens from men), and 7.7% were positive for
C. trachomatis
, with a sensitivity and specificity of 100% upon discrepant analysis. The AMP CT is a sensitive and specific nucleic acid hybridization assay for the detection of
C. trachomatis
in endocervical swab specimens from women, urethral swab specimens from men, and urine specimens from men and women.
Sequence Variation in the Small-Subunit rRNA Gene of
Plasmodium malariae
and Prevalence of Isolates with the Variant Sequence in Sichuan, China
Authors: Liu Qing , Zhu Shenghua , Mizuno Sahoko , Kimura Masatsugu , Liu Peina , Isomura Shin , Wang Xingzhen , Kawamoto Fumihiko
Published: December 2019

By two PCR-based diagnostic methods,
Plasmodium malariae
infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of
P. malariae
have been officially reported for the last 2 decades. In addition, a variant form of
P. malariae
which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of
Plasmodium
sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of
P. vivax
or
P. knowlesi
and its 3′ region is identical to that of
P. malariae
. The same sequence variations were also found in
P. malariae
isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia.
Molecular Characterization of
Bacillus
Strains Involved in Outbreaks of Anthrax in France in 1997
Authors: Patra Guy , Vaissaire Josée , Weber-Levy Martine , Le Doujet Claudine , Mock Michèle
Published: December 2019

Outbreaks of anthrax zoonose occurred in two regions of France in 1997. Ninety-four animals died, and there were three nonfatal cases in humans. The diagnosis of anthrax was rapidly confirmed by bacteriological and molecular biological methods. The strains of
Bacillus anthracis
in animal and soil samples were identified by a multiplex PCR assay. They all belonged to the variable-number tandem repeat (VNTR) group (VNTR)
3
. A penicillin-resistant strain was detected. Nonvirulent bacilli related to
B. anthracis
, of all VNTR types, were also found in the soil.
Comparison of Four Serological Tests To Determine the CagA or VacA Status of
Helicobacter pylori
Strains
Authors: Yamaoka Yoshio , Kodama Tadashi , Graham David Y. , Kashima Kei
Published: December 2019

We compared four tests for antibodies to CagA or VacA, HelicoBlot 2.0, RIDA Blot Helicobacter, CHIRON RIBA
H. pylori
SIA, and an enzyme-linked immunosorbent assay using recombinant CagA. Immunoblot assays were accurate for determining
Helicobacter pylori
status but poor for determining CagA or VacA status (accuracy, 66 to 80% for CagA status and 34 to 67% for VacA status). None can be recommended for determining CagA or VacA status.
Streptococcal Pharyngitis: Impact of a High-Sensitivity Antigen Test on Physician Outcome
Authors: Needham Cynthia A. , McPherson Kenneth A. , Webb Kenneth H.
Published: December 2019

The purpose of the present study was to determine whether the availability of results from a high-sensitivity, rapid test for group A streptococci (Strep A OIA; BioStar, Inc., Boulder, Colo.) improves physician outcome. The study population included 465 consecutive patients with symptoms of acute pharyngitis seen in two outpatient clinics in a large suburban medical center; one clinic, a walk-in clinic (WIC), primarily saw adult patients, and one clinic, a pediatric and adolescent medicine clinic (PED), primarily saw pediatric patients. We measured improvement in physician outcome by comparing physician intent for prescribing an antibiotic based on clinical impression with physician practice once the results of the Strep A OIA were known. Based upon intent, the physicians seeing WIC patients (WIC physicians) would have prescribed an appropriate antibiotic course for 42% of patients with cultures positive for group A beta-hemolytic streptococci (GABHS) and 61% of patients with cultures negative for GABHS. After receiving the results of the Strep A OIA, WIC physicians prescribed an appropriate antibiotic course for 81% of patients with positive cultures and 72% of patients with negative cultures. Based upon intent, the physicians seeing PED patients (PED physicians) would have prescribed an appropriate antibiotic course for 35% of patients with positive cultures and 77% of patients with negative cultures. After receiving the results of the Strep A OIA, PED physicians prescribed an appropriate antibiotic course for 90% of patients with positive cultures and 81% of patients with negative cultures. Based on a 14.5% prevalence of GABHS among WIC patients, Strep A OIA improved the overall WIC physician outcome from 58 to 74%. Based on a 31.5% prevalence of GABHS among PED patients, Strep A OIA improved the PED physician outcome from 64 to 84%. Had Strep A OIA alone guided therapeutic choice, physicians would have prescribed an appropriate antibiotic course for 95% of the patients at the time of the initial encounter. We conclude that the use of Strep A OIA improves physician outcome.
Expanded Diversity among Californian
Borrelia
Isolates and Description of
Borrelia bissettii
sp. nov. (Formerly
Borrelia
Group DN127)
Authors: Postic D. , Ras N. Marti , Lane R. S. , Hendson M. , Baranton G.
Published: December 2019

Up to now, the only species in the complex
Borrelia burgdorferi
sensu lato known to cause Lyme borreliosis in the United States has been
B. burgdorferi
sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of
B. burgdorferi
sensu lato group DN127, we analyzed the nucleotide sequences of the
rrf-rrl
intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American
B. burgdorferi
sensu stricto strains (6 from California). The spacer region sequences from the entire
B. burgdorferi
sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the
B. burgdorferi
sensu lato complex (
B. afzelii
,
B. garinii
,
B. andersonii
,
B. japonica
,
B. burgdorferi
sensu stricto,
B. valaisiana
, and
B. lusitaniae
) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from
B. burgdorferi
sensu stricto. Within this cluster, the deep branches expressing the distances between the
rrf-rrl
sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by
B. burgdorferi
sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the
rrs
sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American
B. burgdorferi
sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species,
B. bissettii
sp. nov., and that other related but distinct strains, which require further characterization, be referred to as
Borrelia
spp.
Clonal Distribution of Methicillin-Resistant
Staphylococcus aureus
in Poland
Authors: Leski T. , Oliveira D. , Trzcinski K. , Sanches I. Santos , de Sousa M. Aires , Hryniewicz W. , de Lencastre H.
Published: December 2019

We report on a study of 158 methicillin-resistant
Staphylococcus aureus
(MRSA) clinical isolates obtained from 1990 to 1996 in 18 different hospitals in Poland. All isolates were recovered from infection and carriage sites of patients, carriage sites of health care personnel, and hospital environment samples. Fifty-seven MRSA strains described here were studied previously and these were divided into two different clusters according to the degree of heterogeneity of methicillin resistance expression. The aim of this study was to extend the correlation between the two clusters and identify the clonal identities among all isolates by a combination of different methodologies: (i) analysis of
mecA
polymorphs and Tn
554
insertion patterns and (ii) determination of pulsed-field gel electrophoresis patterns of chromosomal
Sma
I digests. Ninety-seven of 158 strains showed a heterogeneous expression of resistance to methicillin. Among these, 75 (77.3%) were
Cla
I-
mecA
type I,
Cla
I-Tn
554
type NH (NH, no homology with transposon Tn
554
), and pulsed-field gel electrophoresis (PFGE) pattern A (I::NH::A); 10 isolates were III::B::M (10.3%); and the remaining clones included a few or single isolates. The isolates with homogeneous expression of resistance to methicillin (
n
= 61) were predominantly
Cla
I-
mecA
type III (49 of 61 [80.3%]) but had great variability in their
Cla
I-Tn
554
and PFGE patterns. This study confirmed the existence of two main clusters of MRSA in Poland.
Antipneumococcal Activities of Levofloxacin and Clarithromycin as Determined by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies
Authors: Clark Catherine L. , Jacobs Michael R. , Appelbaum Peter C.
Published: December 2019

The activities of levofloxacin and clarithromycin against 199 penicillin- and macrolide-susceptible and -resistant pneumococci were tested by agar and microdilution methods in air and by disk diffusion and E-test methods in air and CO
2
. For levofloxacin, ≥99.0% of strains were susceptible at ≤2.0 μg/ml with zone diameters of ≥17 mm, regardless of incubation in air or CO
2
. Although zone sizes were smaller and E-test MICs were higher for clarithromycin in CO
2
than those in air, category differences were minor, and susceptibility rates for clarithromycin were similar to those obtained by agar and microdilution in air (range, 76.9 to 80.9% by all methods). For clarithromycin, adjustment of breakpoints based upon distribution of results resulted in susceptibility rates which were similar by all methods (75.8 to 76.9% susceptible, 0 to 1.5% intermediate, 22.6 to 23.1% resistant). Minor discrepancies were obtained with levofloxacin for one strain (0.5%) by microdilution and two strains (1.0%) by disk diffusion in CO
2
. For clarithromycin, minor discrepancies were found in three strains (1.5%) by microdilution, seven strains (3.5%) by agar dilution, four strains (2.0%) by E-test in air, six strains (3.0%) by disk diffusion in air, and five strains (2.5%) by disk diffusion in CO
2
. Major discrepancies occurred with levofloxacin in one strain (0.5%) by microdilution but were not found with clarithromycin. Very major discrepancies were not seen with levofloxacin, but occurred with clarithromycin in five strains (2.5%) by microdilution, three strains (1.5%) by agar dilution, two strains (1.0%) by E-test in air, eight strains (4.0%) by disk diffusion in air, and one strain (0.5%) by disk diffusion in CO
2
.
Pooling of Urine Samples for Screening for
Neisseria gonorrhoeae
by Ligase Chain Reaction: Accuracy and Application
Authors: Kacena Katherine A. , Quinn Sean B. , Hartman Suzanne C. , Quinn Thomas C. , Gaydos Charlotte A.
Published: December 2019

The accuracy of detection of genital
Neisseria gonorrhoeae
infection in pooled urine samples by ligase chain reaction (LCR) was examined in three populations. Firstly, urine specimens from 300 female military recruits (FMR) were tested by LCR individually and in pools of four and six. Secondly, 300 urine specimens from middle-school students (MSS) were tested individually by LCR, and then the processed specimens were stored frozen for subsequent testing in pools of 4 and 10. Thirdly, 600 frozen urine specimens from high-school students (HSS) were tested by using the LCR pooling algorithm, i.e., testing processed specimens in pools of four in one test unit dose, and retesting individual specimens from positive pools. Finally, the pooling algorithm results were compared to culture results for a subset of 344 students from the original 600 HSS from whom cervical or urethral samples were taken at the discretion of the school nurse practitioners. Compared to individual testing of specimens by LCR in the FMR population, the pooling-by-four algorithm was 100% sensitive (5 of 5) and 100% pool specific (70 of 70), and the pool-by-six algorithm was 100% sensitive (5 of 5) and 100% pool specific (45 of 45). In the MSS population, the pool-by-4 algorithm was 95.8% sensitive (23 of 24) and 100% (52 of 52) pool specific, and the pool-by-10 algorithm was 95.8% sensitive (23 of 24) and 100% (17 of 17) pool specific. In the subset of 344 HSS from whom endocervical or urethral specimens were collected for culture, 31 were positive by LCR in urine and 26 were positive by culture. After results discrepant between culture and LCR were adjudicated by a confirmatory LCR test, the pooling algorithm was 93.8% (30 of 32) sensitive and 99.7% (311 of 312) specific. Culture from these 344 HSS was 81.3% (26 of 32) sensitive. The pooling algorithm reduced the cost of the
N. gonorrhoeae
LCR assay by 60% compared to individual testing of the HSS specimens and was both sensitive and specific.
Human Cytomegalovirus (HCMV) Encephalitis in an Immunocompetent Young Person and Diagnostic Reliability of HCMV DNA PCR Using Cerebrospinal Fluid of Nonimmunosuppressed Patients
Authors: Prösch Susanna , Schielke Eva , Reip Angela , Meisel Helga , Volk Hans-Dieter , Einhäupl Karl M. , Krüger Detlev H.
Published: December 2019

Human cytomegalovirus (HCMV) encephalitis in adult nonimmunosuppressed patients has rarely been reported. We have diagnosed HCMV encephalitis in an anti-HCMV immunoglobulin G-negative, nonimmunosuppressed young woman by HCMV DNA PCR and virus isolation from cerebrospinal fluid (CSF). At the same time, HCMV antigen and HCMV DNA could be demonstrated in peripheral blood leukocytes, and the virus was isolated in fibroblast cultures. After 22 days of acute illness, the virus disappeared from the CSF. Remarkably, the patient did not generate detectable anti-HCMV antibodies within 5 months after the beginning of illness. To investigate the significance of HCMV DNA detection in CSF, samples of CSF, blood cells, and serum from 35 nonimmunosuppressed patients with various neurological disorders (but no herpes simplex virus central nervous system [CNS] disease) were tested for HCMV DNA, antigen, and antibodies. Eleven of these patients were found to be positive for virus DNA and/or antigen in peripheral blood leukocytes. Additionally, HCMV DNA was detected in the CSF of two patients with noninflammatory CNS diseases. A causative role of HCMV in the CNS diseases of these two patients was not evident. In summary, HCMV DNA amplification from CSF samples is a very suitable method to verify HCMV-associated encephalitis, but it should be taken into consideration that there are few cases of positive PCR with DNA from CSF without any known clinical correlative.
Rapid Assay for Simultaneous Detection and Differentiation of Immunoglobulin G Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2
Authors: Vallari Ana S. , Hickman Robert K. , Hackett John R. , Brennan Catherine A. , Varitek Vincent A. , Devare Sushil G.
Published: December 2019

A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.
Comparison of Phenotypic and Genotypic Techniques for Identification of Unusual Aerobic Pathogenic Gram-Negative Bacilli
Authors: Tang Yi-Wei , Ellis Nicole M. , Hopkins Marlene K. , Smith Douglas H. , Dodge Deborah E. , Persing David H.
Published: December 2019

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (
P
= 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (
P
= 0.005), respectively. Four
Acinetobacter
and three
Bordetella
isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.
Effect of Serial Passage in Tissue Culture on Sequence ofomp1fromChlamydia trachomatisClinical Isolates
Authors: Stothard Diane R. , Van Der Pol Barbara , Smith Nancy J. , Jones Robert B.
Published: December 2019

Previous epidemiological studies ofChlamydia trachomatisfrequently have required expansion of isolates in tissue culture. The possibility thatC. trachomatis omp1might undergo mutation during such expansion has not been examined systematically. We found no differences in theomp1sequences from 10 clinical specimens before and after 20 in vitro passages.
Rapid PCR Detection of
Helicobacter pylori
-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material
Authors: Björkholm Britta , Befrits Ragnar , Jaup Bernhard , Engstrand Lars
Published: December 2019

We have developed a PCR-based method to detect macrolide resistance and the virulence gene
cagA
in
Helicobacter pylori
within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.
Isolation of Blood-Borne
Mycobacterium avium
by Using the Nonradioactive BACTEC 9000 MB System and Comparison with a Solid-Culture System
Authors: Jacomo Véronique , Musso Didier , Gevaudan Marie-José , Drancourt Michel
Published: December 2019

We conducted a 12-month prospective study comparing two approaches to the detection of
Mycobacterium avium
in the blood of human immunodeficiency virus type 1-infected patients, namely, a lytic centrifugation system combined with Middlebrook solid culture medium (the conventional procedure) and the nonradiometric BACTEC 9000 MB system. Species identification relied on 16S rRNA probe hybridization and cell wall fatty acids chromatography.
M. avium
was isolated in 17 of 345 (5%) blood specimens by the BACTEC 9000 MB automated system and in 14 of 345 (4%) blood specimens by the conventional procedure (nonsignificant, χ
2
test). Detection time was 16 ± 6 days by the BACTEC 9000 MB automated system and 27 ± 3 days by the conventional procedure (P < 0.001, Student t test). Non-
M. avium
mycobacteria were not recovered during the study period. Contamination rate was 8% (30 specimens) by the BACTEC 9000 MB system and 0% by the conventional procedure, indicating the necessity of using an antibiotic mixture (PANTA, consisting of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin). Working time was 1 min 30 s by the BACTEC 9000 MB system and 8 min by the conventional procedure, which was 1.8 times more expensive than the BACTEC system. Use of the BACTEC 9000 MB system increased the sensitivity of
M. avium
detection and reduced detection time in blood culture.
Detection of Bovine Herpesvirus 1 Glycoprotein E Antibodies in Individual Milk Samples by Enzyme-Linked Immunosorbent Assays
Authors: Wellenberg G. J. , Verstraten E. R. A. M. , Mars M. H. , Van Oirschot J. T.
Published: December 2019

The purpose of this study was to determine whether individual milk samples can replace serum samples for the detection of bovine herpesvirus 1 (BHV1) glycoprotein E (gE)-specific antibodies. Serum and milk samples were collected at the same time from cattle in BHV1-free herds, cattle in unvaccinated herds, and cattle in herds that were vaccinated twice with a BHV1 marker vaccine. The samples were tested in two gE enzyme-linked immunosorbent assay (ELISA) systems. In comparison to serum, the results showed that the gE-blocking ELISA was highly sensitive for testing milk samples (0.96). In contrast, the gE ELISA was less sensitive (0.79). The specificities of the gE-blocking ELISA and the gE ELISA for testing milk samples were very high (1.00 and 0.99, respectively). The presented results indicate that individual milk samples, which can be collected relatively easily and inexpensively, can be used instead of individual serum samples in the gE-blocking ELISA for the screening of cattle for BHV1 gE antibodies.
Detection ofTritrichomonas foetusby PCR and DNA Enzyme Immunoassay Based on rRNA Gene Unit Sequences
Authors: Felleisen Richard S. J. , Lambelet Natacha , Bachmann Philipp , Nicolet Jacques , Müller Norbert , Gottstein Bruno
Published: December 2019

Tritrichomonas foetusis the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken forT. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units ofT. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a singleT. foetusorganism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a directT. foetusscreening test with diagnostic samples.
Amplification of Full-Length Hepatitis B Virus Genomes from Samples from Patients with Low Levels of Viremia: Frequency and Functional Consequences of PCR-Introduced Mutations
Authors: Günther Stephan , Sommer Gunhild , Von Breunig Franziska , Iwanska Alicja , Kalinina Tatyana , Sterneck Martina , Will Hans
Published: December 2019

To facilitate the investigation of hepatitis B virus (HBV) sequence variation, we recently established a method for functional analysis of PCR-amplified full-length HBV genomes. This study aimed at estimating the number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-type HBV DNA template molecules in concentrations like those present in samples from patients with very low levels of viremia were amplified, sequenced (30 kb total), and functionally tested. We found thatTaqpolymerase and aTaq-Pwopolymerase mixture introduced an average of 5.7 and 3.1 mutations per genome, respectively, corresponding to polymerase error rates of 12.1 × 10−5and 6.0 × 10−5. One of 8 genomes (12%) amplified withTaqpolymerase, but 7 of 17 genomes amplified withTaq-Pwopolymerases (41%), remained replication competent. All replication-competent genomes expressed HBs and HBe antigens and had an average of only 0.9 mutations per genome. In contrast, replication-defective genomes had an average of 5.4 mutations, which frequently also disturbed viral antigen expression. From these data we conclude that many of the replication-competent HBV genomes from a clinical specimen will retain their replication and antigen expression phenotypes even after extensive amplification withTaq-Pwopolymerases. Because replication competence is highly sensitive to random mutations, it is the best marker for the identification of HBV genomes with few or no PCR-introduced mutations.
Oligonucleotide Ligation Assay for Detecting Mutations in the Human Immunodeficiency Virus Type 1
pol
Gene That Are Associated with Resistance to Zidovudine, Didanosine, and Lamivudine
Authors: Edelstein Reva E. , Nickerson Deborah A. , Tobe Vincent O. , Manns-Arcuino Laura A. , Frenkel Lisa M.
Published: December 2019

This report describes the detection of mutations in the
pol
gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the
pol
gene amplified by PCR. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system.