bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Cytomegalovirus Immediate-Early mRNA Detection by Nucleic Acid Sequence-Based Amplification as a New Parameter for Preemptive Therapy in Bone Marrow Transplant Recipients
Authors: Gerna Giuseppe , Baldanti Fausto , Lilleri Daniele , Parea Maurizio , Alessandrino Emilio , Pagani Ambrogio , Locatelli Franco , Middeldorp Jaap , Revello M. Grazia
Published: December 2019

Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of immediate-early (IE) mRNA by nucleic acid sequence-based amplification (NASBA) in a series of 51 bone marrow transplant (BMT) recipients. The qualitative results for IE mRNA obtained by NASBA were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in blood (DNAemia) by PCR as well as by qualitative determination of late pp67 mRNA by NASBA. On the whole, of the 39 HCMV-positive patients (all asymptomatic), HCMV was detected in 14 (35.9%) by quantitation of viremia, 15 (38.5%) by detection of pp67 mRNA by NASBA, 32 (82.1%) by quantitation of DNAemia, and 33 (84.6%) by quantitation of antigenemia, while HCMV was detected in 38 (97.4%) patients by detection of IE mRNA by NASBA. In the immunocompetent host, IE mRNA was not detected by NASBA in 100 blood donors or during reactivated infections in 30 breast-feeding mothers. Likewise, NASBA did not detect IE mRNA in 56 solid-organ transplant recipients in the first 21 days after transplantation. By using NASBA for detection of IE mRNA as the reference standard for detection of HCMV infection in blood samples, the diagnostic sensitivities were 67.7% for quantitation of DNAemia, 59.0% for quantitation of antigenemia, 18.3% for detection of pp67 mRNA by NASBA, and 16.0% for quantitation of viremia. Specificities and negative and positive predictive values were >90.0, >70.0, and >80.0%, respectively, for all four assays. The mean times to first HCMV detection after bone marrow transplantation were 37.7 ± 15.4 days for detection of IE mRNA by NASBA, 39.6 ± 15.6 days for quantitation of antigenemia, 40.9 ± 15.2 days for quantitation of DNAemia, and 43.7 ± 16.3 or 43.7 ± 17.5 days for quantitation of viremia and detection of pp67 mRNA by NASBA, respectively. On the whole, 31 BMT recipients received preemptive therapy by using confirmed antigenemia positivity as a cutoff, while 35 patients could have been treated by using NASBA positivity as a cutoff and 31 could have been treated by using quantitation of DNAemia as a cutoff. In single patients, IE mRNA was detected in every episode of active HCMV infection, mostly preceding and sometimes accompanying antigenemia and DNAemia, whereas pp67 mRNA was detected only concomitantly with the highest peaks of infection. HCMV IE mRNA detection may represent a useful parameter for initiation of preemptive therapy in BMT recipients.
Direct Molecular Typing of
Borrelia burgdorferi
Sensu Lato Species in Synovial Samples from Patients with Lyme Arthritis
Authors: Jaulhac B. , Heller R. , Limbach F. X. , Hansmann Y. , Lipsker D. , Monteil H. , Sibilia J. , Piémont Y.
Published: December 2019

Since Lyme arthritis was first described in the United States, it has now been reported in many countries of Europe. However, very few strains of the causative bacterium,
Borrelia burgdorferi
, have been isolated from synovial samples. For this reason, different molecular direct typing methods were developed recently to assess which species could be involved in Lyme arthritis in Europe. We developed a simple oligonucleotide typing method with PCR fragments from the flagellin gene of
B. burgdorferi
sensu lato, which is able to differentiate seven different
Borrelia
species. Among 10 consecutive PCR-positive patients with Lyme arthritis from the northeastern France, two species were identified in synovial samples:
B. burgdorferi
sensu stricto in 9 cases and
B. garinii
in 1 case. Conversely, all
B. burgdorferi
sensu lato species detected in 10 consecutive PCR-positive biopsies from a second set of patients with erythema migrans from the same geographical area were identified as either
B. afzelii
or
B. garinii
(
P
< 0.001). These results indicate that
B. burgdorferi
sensu stricto is the principal but not the only
Borrelia
species involved in Lyme arthritis in northeastern France.
Technical Assessment of the Affymetrix Yeast Expression GeneChip YE6100 Platform in a Heterologous Model of Genes That Confer Resistance to Antimalarial Drugs in Yeast
Authors: Nau Martin E. , Emerson Lyndal R. , Martin Rodger K. , Kyle Dennis E. , Wirth Dyann F. , Vahey Maryanne
Published: December 2019

The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization. At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms. The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine. Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis.
Coinfection with Multiple TT Virus Strains Belonging to Different Genotypes Is a Common Event in Healthy Brazilian Adults
Authors: Niel Christian , Saback Felipe L. , Lampe Elisabeth
Published: December 2019

Testing of the DNA of TT virus (TTV) was done with serum samples obtained from 191 persons working in a public hospital of the city of Rio de Janeiro, Brazil. TTV DNA was detected by PCR in the sera of 125 (65.4%) individuals. PCR products were cloned, and sequences with a length of 159 bases surrounding the TATA signal region were determined for 100 clones derived from 31 individuals. One clone from each of 23 subjects was sequenced, while 7 to 19 clones from eight individuals were sequenced. None of the sera contained a viral sequence identical to that of any other individual. Phylogenetic analysis revealed the existence of a divergent TTV genotype possessing a single-base deletion at position 140. Among the eight persons for whom various sequences were analyzed, six were coinfected with between two and seven TTV strains belonging to different genotypes. The results suggest that coinfection with multiple TTV strains belonging to different genotypes is a common event in healthy Brazilian adults.
Acute Cerebral Phaeohyphomycosis due to
Wangiella dermatitidis
Accompanied by Cerebrospinal Fluid Eosinophilia
Authors: Chang Chulhun Ludgerus , Kim Dae-Seong , Park Dong June , Kim Hak Jin , Lee Chang Hun , Shin Jong Hee
Published: December 2019

We report a case of cerebral phaeohyphomycosis due to
Wangiella dermaitidis
in an immunocompetent adult man. His cerebrospinal fluid (CSF) showed pleocytosis with a high eosinophil count but without peripheral blood eosinophilia. The present case suggested that this black yeast-like fungus should be included when the causes of CSF eosinophilia are considered, even though it is an extremely rare pathogen.
Evidence from a Nine-Year Birth Cohort Study in Japan of Transmission Pathways of
Helicobacter pylori
Infection
Authors: Malaty Hoda M. , Kumagai Toshiko , Tanaka Eiji , Ota Hiroyoshi , Kiyosawa Kendo , Graham David Y. , Katsuyama Tsutomu
Published: December 2019

We examined the longitudinal changes of
Helicobacter pylori
infection within 46 families with children and 48 couples without children living in Japan. The study cohort was monitored from 1986 to 1994.
H. pylori
status was assessed by the presence of anti-
H. pylori
immunoglobulin G antibodies. At study entry,
H. pylori
prevalence in children with positive mothers was 23% versus 5% in children with negative mothers (odds ratio = 5.3; 95% confidence interval = 0.6 to 42.8). Seroconversion (rate of 1.5%/year) was evident only among children living with positive mothers and did not differ among adults living with or without children. These data strongly support the cluster phenomenon of
H. pylori
infection among families, the key role of the infected mothers in the transmission within families, and the importance of adult-child transmission and not vice versa.
Evaluation of the MB/BacT Mycobacterium Detection System for Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Díaz-Infantes María S. , Ruiz-Serrano María J. , Martínez-Sánchez Lucía , Ortega Arturo , Bouza Emilio
Published: December 2019

The MB/BacT mycobacterium detection system was evaluated for its performance in the susceptibility testing of
Mycobacterium tuberculosis
. Eighty-three
M. tuberculosis
isolates were processed. Results for all isoniazid-, rifampin- and streptomycin-susceptible, isoniazid-resistant, and rifampin-resistant
M. tuberculosis
isolates with the MB/BacT system agreed 100% with those obtained by the agar proportion method. The agreements between the two methods for streptomycin- and ethambutol-resistant isolates were 96.4 and 90.4%, respectively. The susceptibility test results were obtained in 7 days, on average. These data demonstrate that the MB/BacT system is an accurate, nonradiometric method for rapid susceptibility testing of
M. tuberculosis
.
Bartonella henselae
Infection as a Cause of Fever of Unknown Origin
Authors: Tsukahara Masato , Tsuneoka Hidehiro , Iino Hidechika , Murano Ichiro , Takahashi Hirohiko , Uchida Masashi
Published: December 2019

Fourteen of 41 patients (34%) with a serological diagnosis of
Bartonella henselae
infection were found to have prolonged fever or fever of unknown origin, suggesting that generalized systemic
B. henselae
infection is not rare in immunocompetent healthy individuals.
Multiplex PCRs for Identification of
Escherichia coli
Virulence Genes
Authors: Pass M. A. , Odedra R. , Batt R. M.
Published: December 2019

PCRs were developed to detect 11
Escherichia coli
virulence genes. Primers amplified the respective genes without cross-reaction with other genes. Specificity was maintained in multiplex reactions; excellent amplification of target genes was possible with a minimum of four multiplex reactions. These reactions successfully identified genes in
E. coli
from the feces of four dogs.
An Important Proportion of Genital Samples Submitted for
Chlamydia trachomatis
Detection by PCR Contain Small Amounts of Cellular DNA as Measured by β-Globin Gene Amplification
Authors: Coutlée François , de Ladurantaye Manon , Tremblay Carole , Vincelette Jean , Labrecque Louise , Roger Michel
Published: December 2019

We assessed the quality of genital samples submitted for
Chlamydia trachomatis
detection by PCR by a second PCR assay for the presence of human β-globin DNA. Endocervical and urethral samples were first tested by the COBAS AMPLICOR
C. trachomatis
assay (Roche Diagnostic Systems) with an internal control and were then amplified for the presence of β-globin DNA with primers PC04 and GH20. Samples that contained inhibitors were retested after dilution 1:10. A total of 407 genital samples (311 endocervical swabs from 311 women and 96 urethral swabs from 95 men and 1 woman) collected over a 1-month period were evaluated. The internal control could not be amplified, despite dilution, from 3 of 23 samples that were retested after dilution because of inhibition, leaving 404 samples that could be analyzed by PCR. Eleven samples tested positive for
C. trachomatis
. Thirty (7.4%) of the 404 samples were negative for β-globin. Twelve of the 23 undiluted samples that contained inhibitors tested positive for β-globin DNA. Amplification of β-globin DNA in samples submitted for
C. trachomatis
detection by the COBAS AMPLICOR
C. trachomatis
assay demonstrated that an important proportion of the samples did not contain cellular DNA. Assessment of the quality of the samples for PCR analysis by β-globin amplification is feasible but cannot replace use of the internal control.
A 5′ Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the
mecA
Gene in Staphylococci
Authors: Killgore George E. , Holloway Brian , Tenover Fred C.
Published: December 2019

In an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional PCR results to the results generated using the TaqMan 5′ nuclease PCR kit in conjunction with an ABI Prism 7700 Sequence Detector for detecting the
mecA
gene in various species of staphylococci. DNA was extracted using two techniques. The first used a high-salt extraction method suitable for conventional PCR but resulted in a 7.2% rate of PCR inhibition with the TaqMan technique. PCR inhibition could be overcome by diluting samples 1:5 prior to testing. The second method used the Qiagen QIAamp Tissue Kit; no instances of PCR inhibition were encountered with this method. A total of 197 (96%) of the 206 samples with no inhibition showed agreement between the two methods. Eight of the nine disagreements were likely the result of low-level DNA cross contamination caused by frequent specimen handling. Target DNA in all eight of these samples was first detected in the initial tests only after >30 PCR cycles, and all were negative upon repeat testing even after 40 PCR cycles using freshly extracted DNA. Among those positive samples in agreement, target DNA was invariably detected before 30 PCR cycles. The TaqMan assay eliminated the need to load, run, stain, and read agarose gels and provided the advantage of instant detection of PCR product by laser-activated fluorescence. Thus, final results were obtained 2 h after PCR was initiated, as opposed to a requirement of 2 days to examine 96 samples by agarose gel electrophoresis.
Real-Time Automated PCR for Early Diagnosis and Monitoring of Cytomegalovirus Infection after Bone Marrow Transplantation
Authors: Machida Utako , Kami Masahiro , Fukui Takafumi , Kazuyama Yukumasa , Kinoshita Moritoshi , Tanaka Yuji , Kanda Yoshinobu , Ogawa Seishi , Honda Hiroaki , Chiba Shigeru , Mitani Kinuko , Muto Yoshitomo , Osumi Kazuoki , Kimura Satoshi , Hirai Hisamaru
Published: December 2019

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 10
7
copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.
Comparison of the Serodia
Treponema pallidum
Particle Agglutination, Captia Syphilis-G, and SpiroTek Reagin II Tests with Standard Test Techniques for Diagnosis of Syphilis
Authors: Pope Victoria , Fears Martha B. , Morrill William E. , Castro Arnold , Kikkert Susan E.
Published: December 2019

We compared the microhemagglutination assay for
Treponema pallidum
(MHA-TP), a treponemal test, with two other treponemal tests, the Serodia
Treponema pallidum
particle agglutination (TP-PA) assay and the Captia Syphilis-G enzyme immunoassay, using 390 clinical serum samples. We also compared two nontreponemal tests, the rapid plasma Reagin (RPR) card test and the SpiroTek Reagin II test. Agreements of the MHA-TP with the TP-PA test and the Syphilis-G test were 97.4 and 97.7%, respectively. There was 89.2% agreement between the RPR and Reagin II tests. The Reagin II test was more apt to be reactive if the treponemal test was also reactive. We conclude that either the Serodia TP-PA test or the Captia Syphilis-G test is an appropriate substitute for the MHA-TP and that the Spirotek Reagin II test could substitute for the RPR test as a screening test.
Stability of IS
6110
Restriction Fragment Length Polymorphism Patterns of
Mycobacterium tuberculosis
Strains in Actual Chains of Transmission
Authors: Niemann Stefan , Rüsch-Gerdes Sabine , Richter Elvira , Thielen Heiko , Heykes-Uden Helga , Diel Roland
Published: December 2019

The stability of IS
6110
restriction fragment length polymorphism (RFLP) patterns of
Mycobacterium tuberculosis
strains in actual transmission chains has been assessed by analyzing the variability of IS
6110
RFLP patterns of strains in fingerprint clusters that have been confirmed by classical epidemiological data. Forty susceptible and 35 drug-resistant (including 17 multidrug-resistant)
M. tuberculosis
strains obtained from 75 patients living in Germany have been analyzed. The epidemiological relationship among strains within the fingerprint clusters has been verified by family contacts (14 clusters) or by contact tracing of the public health offices (7 clusters). The time spans between the first and the last isolate of one cluster ranged from less than 1 to 29 months. Of the 75 strains only 1 showed a one-band variation when compared to the other nine isolates grouped in the same cluster, corresponding with a rate of change of ∼1.9% per possible transmission (one index patient per cluster was subtracted from the total number of isolates). These results confirm a high degree of stability of IS
6110
RFLP patterns of transmitted
M. tuberculosis
strains. Furthermore, the data presented indicate that isolates with identical IS
6110
DNA fingerprint patterns are a good indicator for the rate of recent transmission in a study population.
Comparison of 16S rRNA Gene PCR and BACTEC 9240 for Detection of Neonatal Bacteremia
Authors: Jordan J. A. , Durso M. B.
Published: December 2019

Ten percent of infants born in the United States are admitted to neonatal intensive care units (NICU) annually. Approximately one-half of these admissions are from term infants (>34 weeks of gestation) at risk for systemic infection. Most of the term infants are not infected but rather have symptoms consistent with other medical conditions that mimic sepsis. The current standard of care for evaluating bacterial sepsis in the newborn is performing blood culturing and providing antibiotic therapy while awaiting the 48-h preliminary result of culture. Implementing a more rapid means of ruling out sepsis in term newborns could result in shorter NICU stays and less antibiotic usage. The purpose of this feasibility study was to compare the utility of PCR to that of conventional culture. To this end, a total of 548 paired blood samples collected from infants admitted to the NICU for suspected sepsis were analyzed for bacterial growth using the BACTEC 9240 instrument and for the bacterial 16S rRNA gene using a PCR assay which included a 5-h preamplification culturing step. The positivity rates by culture and PCR were 25 (4.6%) and 27 (4.9%) positive specimens out of a total of 548 specimens, respectively. The comparison revealed sensitivity, specificity, and positive and negative predictive values of 96.0, 99.4, 88.9, and 99.8%, respectively, for PCR. In summary, this PCR-based approach, requiring as little as 9 h of turnaround time and blood volumes as small as 200 μl, correlated well with conventional blood culture results obtained for neonates suspected of having bacterial sepsis.
Simultaneous Detection and Genotyping of “Norwalk-Like Viruses” by Oligonucleotide Array in a Reverse Line Blot Hybridization Format
Authors: Vinjé Jan , Koopmans Marion P. G.
Published: December 2019

“Norwalk-like viruses” (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.
Virulent Rough Filaments of
Listeria monocytogenes
from Clinical and Food Samples Secreting Wild-Type Levels of Cell-Free p60 Protein
Authors: Rowan Neil J. , Candlish Alan A. G. , Bubert Andreas , Anderson John G. , Kramer Karl , McLauchlin Jim
Published: December 2019

Atypical rough cell filaments of
Listeria monocytogenes
(designated FR variants), isolated from clinical and food samples, form long filaments up to 96 μm in length and demonstrated wild-type levels of adherence, invasion, and cytotoxicity to human epithelial HEp-2, Caco-2, and HeLa cells. Unlike previously described avirulent rough mutants of
L. monocytogenes
that secrete diminished levels of the major extracellular protein p60 and that form long chains that consist of multiple cells of similar size (designated MCR variants), FR variants secreted wild-type or greater levels of p60. This study shows that virulent filamentous forms of
L. monocytogenes
occur in clinical and food environments and have atypical morphological characteristics compared to those of the wild-type form.
Assessment of Viral Loads in Patients with Chronic Hepatitis C with AMPLICOR HCV MONITOR Version 1.0, COBAS HCV MONITOR Version 2.0, and QUANTIPLEX HCV RNA Version 2.0 Assays
Authors: Martinot-Peignoux Michéle , Le Breton Véronique , Fritsch Sandrine , Le guludec Gaëlle , Labouret Nathalie , Keller Françoise , Marcellin Patrick
Published: December 2019

The correlation between response to antiviral therapy and pretreatment viral load in patients with chronic hepatitis C has prompted the development of quantitative assays to measure viral load. The aim of our study was to assess the clinical relevance of the newly developed semiautomated PCR system COBAS HCV MONITOR version 2.0 in comparison with (i) the AMPLICOR HCV MONITOR version 1.0 assay, which underestimates RNA concentration of hepatitis C virus (HCV) genotypes 2 to 6, and (ii) the QUANTIPLEX HCV RNA version 2.0 assay, which achieves equivalent quantification for each HCV genotype, with samples from 174 patients diagnosed with chronic hepatitis C before therapy. The level and range of quantification measured with AMPLICOR HCV MONITOR version 1.0 were 1 log lower than when measured with the COBAS HCV MONITOR version 2.0, at 0.261 × 10
6
RNA copies/ml (range, 0.001 × 10
6
to 2.50 × 10
6
RNA copies/ml) and 4.032 × 10
6
RNA copies/ml (range, 0.026 × 10
6
to 72.6 × 10
6
RNA copies/ml), respectively. The two assays showed a poor correlation (
r
2
= 0.175). The level and range of quantification were similar when measured with the COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays, at 3.03 × 10
6
RNA copies/ml (range, 0.023 × 10
6
to 72.6 × 10
6
RNA copies/ml) and 4.91 Meq/ml (range, 0.200 to 49.5 Meq/ml), respectively. The two assays showed a strong correlation (
r
2
= 0.686) for each HCV genotype. The duration of treatment (6 or 12 months) is modulated according to HCV genotype and viral load. Our results indicate that COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays showing an equal dynamic range for each HCV genotype are suitable tools to assess patients before therapy.
Early Virological Failure in Naive Human Immunodeficiency Virus Patients Receiving Saquinavir (Soft Gel Capsule)-Stavudine-Zalcitabine (MIKADO Trial) Is Not Associated with Mutations Conferring Viral Resistance
Authors: Mouroux M. , Yvon-Groussin A. , Peytavin G. , Delaugerre C. , Legrand M. , Bossi P. , Do B. , Trylesinski A. , Diquet B. , Dohin E. , Delfraissy J. F. , Katlama C. , Calvez V. ,   The MIKADO Study Group
Published: December 2019

The MIKADO trial was designed to evaluate the efficacy of stavudine-zalcitabine-saquinavir (soft gel capsule) [d4T-ddC-SQV(SGC)] in 36 naive patients (−3.3 log10units at week 24 [W24]). Among the 29 patients remaining on d4T-ddC-SQV(SGC) until W24, 10 harbored a virological failure (viral load of >200 copies/ml at W24) (group 1). To determine the reasons for therapeutic failure, genotypic and phenotypic resistance test results and SQV concentrations in plasma were analyzed and compared to those in successfully treated patients (viral load of <200 copies/ml at W24) (group 2). Reverse transcriptase and protease genotypic analyses in group 1 revealed the acquisition of only one SQV-associated mutation (L90M) in only two patients. There was no significant increase in the 50 or 90% inhibitory concentration of SQV in patients with or without the L90M mutation. However, the fact that two patients developed an L90M mutation only 4 weeks after relapse points to the need for genotypic resistance testing in the context of an initial failure of the antiretroviral regimen. At W24, the median SQV concentration in group 1 (71 ng/ml) was significantly lower than in group 2 (475 ng/ml), and the plasma SQV concentration was correlated with the viral load at W24 (r= −0.5;P< 0.05) and with the drop in viral load between day 0 and W24 (r= −0.5;P< 0.01). These results and the fact that the plasma SQV concentrations in the two groups prior to relapse (W12) were not significantly different strongly suggest that the early failure of this combination is not due to viral resistance but to a lack of compliance, pharmacological variability, and drug interactions or a combination of these factors.
Rapid Diagnosis of Legionnaires' Disease Using an Immunochromatographic Assay for
Legionella pneumophila
Serogroup 1 Antigen in Urine during an Outbreak in The Netherlands
Authors: Wever Peter C. , Yzerman Ed P. F. , Kuijper Ed J. , Speelman Peter , Dankert Jacob
Published: December 2019

A new immunochromatographic assay for rapid qualitative detection of
Legionella pneumophila
serogroup 1 antigen in urine specimens was used during an outbreak of legionellosis in The Netherlands. The assay seems of the utmost value in providing a rapid diagnosis of Legionnaires' disease in patients with severe community-acquired pneumonia in an outbreak setting.
Rapid Differentiation of
Borrelia garinii
from
Borrelia afzelii
and
Borrelia burgdorferi
Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the
recA
Gene
Authors: Pietilä Johanna , He Qiushui , Oksi Jarmo , Viljanen Matti K.
Published: December 2019

To differentiate the
Borrelia burgdorferi
sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial
recA
gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from
B. burgdorferi
reference strains and 16 were isolated from
B. burgdorferi
strains cultured from
Ixodes ricinus
ticks; 13 were directly isolated from nine human biopsy specimens and four
I. ricinus
tick midguts. The melting temperature of
B. garinii
was 2°C lower than that of
B. burgdorferi
sensu stricto and
B. afzelii
. Melting curve analysis offers a rapid alternative for identification and detection of
B. burgdorferi
sensu lato genospecies.
Immunoglobulin A1 Protease Activity inGemella haemolysans
Authors: Lomholt Jeanet A. , Kilian Mogens
Published: December 2019

The purpose of this study was to determine the occurrence and nature of immunoglobulin A1 (IgA1) protease activity in members of the genusGemellaand related taxa. Among a total of 22Gemellastrains belonging to the four speciesGemella haemolysans,Gemella morbillorum,Gemella sanguinis, andGemella bergeriaeand four reference strains of the speciesHelcococcus kunzii,Facklamia hominis, andGlobicatella sanguinis, IgA1 protease activity was an exclusive character of all nine isolates ofG. haemolysans. The IgA1 protease ofG. haemolysansappears to be a metallo-type IgA1 protease that cleaves the Pro227-Thr228peptide bond in the hinge region of the α1 chain like that of severalStreptococcusspecies. Phenotypic characterization of the isolates demonstrates that screening for IgA1 protease activity provides a valuable means for species differentiation in this group of bacteria.
Surveillance of Rotavirus Strains in the United States: Identification of Unusual Strains
Authors: Griffin D. D. , Kirkwood C. D. , Parashar U. D. , Woods P. A. , Bresee J. S. , Glass R. I. , Gentsch J. R. ,   the National Rotavirus Strain Surveillance System Collaborating Laboratories
Published: December 2019

Rotavirus strains from 964 fecal specimens collected from children at 11 U.S. hospital laboratories from November 1997 to March 1998 and from samples collected at 12 laboratories from November 1998 to March 1999 were typed for G and P proteins. Serotype G1 was the predominant serotype in 1997–1998 (88%), followed by G2 (6.2%), G9 (3.3%), and G3 (1.5%). This pattern was similar to that seen in 1998–1999: G1 (79%), G2 (15%), G9 (3.0%), G4 (1.6%), and G3 (0.3%). Novel P[9] strains were identified in both seasons, and analysis of a 364-nucleotide fragment from gene segment 4 of one of the strains demonstrated 97.3% nucleotide identity with the prototype P3[9],G3 strain, AU1, isolated in Japan. This is the first report of a human AU1-like strain in the United States. These results reinforce our initial findings that serotype G9 persists in the United States but has not become a predominant strain and that the common serotypes G1 to G4 account for almost 90% of strains in circulation. Other uncommon strains exist in the United States but may have been overlooked before because of their low prevalence and the use of inadequate diagnostic tools.
Rapid Automated Antimicrobial Susceptibility Testing of
Streptococcus pneumoniae
by Use of the bioMerieux VITEK 2
Authors: Jorgensen James H. , Barry Arthur L. , Traczewski M. M. , Sahm Daniel F. , McElmeel M. Leticia , Crawford Sharon A.
Published: December 2019

The VITEK 2 is a new automated instrument for rapid organism identification and susceptibility testing. It has the capability of performing rapid susceptibility testing of
Streptococcus pneumoniae
with specially configured cards that contain enriched growth medium and antimicrobial agents relevant for this organism. The present study compared the results of testing of a group of 53 challenge strains of pneumococci with known resistance properties and a collection of clinical isolates examined in two study phases with a total of 402 and 416 isolates, respectively, with a prototype of the VITEK 2. Testing was conducted in three geographically separate laboratories; the challenge collection was tested by all three laboratories, and the unique clinical isolates were tested separately by the individual laboratories. The VITEK 2 results of tests with 10 antimicrobial agents were compared to the results generated by the National Committee for Clinical Laboratory Standards reference broth microdilution MIC test method. Excellent interlaboratory agreement was observed with the challenge strains. The overall agreement within a single twofold dilution of MICs defined by the VITEK 2 and reference method with the clinical isolates was 96.3%, although there were a number of off-scale MICs that could not be compared. The best agreement with the clinical isolates was achieved with ofloxacin and chloramphenicol (100%), and the lowest level of agreement among those drugs with sufficient on-scale MICs occurred with trimethoprim-sulfamethoxazole (89.7%). Overall there were 1.3% very major, 6.6% minor, and no major interpretive category errors encountered with the clinical isolates, although >80% of the minor interpretive errors involved only a single log
2
dilution difference. The mean time for generation of susceptibility results with the clinical isolates was 8.1 h. The VITEK 2 provided rapid, reliable susceptibility category determinations with both the challenge and clinical isolates examined in this study.
Nationwide German Multicenter Study on Prevalence of Antibiotic Resistance in Staphylococcal Bloodstream Isolates and Comparative In Vitro Activities of Quinupristin-Dalfopristin
Authors: von Eiff Christof , Reinert Ralf René , Kresken Michael , Brauers Johannes , Hafner Dieter , Peters Georg
Published: December 2019

Antibiotic-resistant gram-positive bacteria have become an increasing problem in the last two decades. In order to evaluate the prevalence of antibiotic resistance in staphylococcal bloodstream isolates in Germany, 2,042 staphylococci collected in 21 tertiary-care hospitals were investigated during a 3-year period (March 1996 to March 1999). Altogether, 1,448
S. aureus
isolates and 594 coagulase-negative staphylococci (CoNS) that comprised 13 different species were included. Furthermore, the antistaphylococcal activities of quinupristin-dalfopristin were compared with those of eight other compounds by the broth microdilution method. The rates of oxacillin resistance in
Staphylococcus aureus
,
S. epidermidis
,
S. haemolyticus
, and other CoNS were 13.5, 69, 90, and 34%, respectively. In oxacillin-resistant strains high rates of resistance (up to 100%) to erythromycin, clindamycin, ciprofloxacin, and gentamicin were also observed. However, no strain appeared to be resistant to vancomycin or quinupristin-dalfopristin. The streptogramin combination exhibited excellent in vitro activity against all staphylococcal species tested, regardless of the patterns of resistance to other drug classes. In terms of MICs at which 90% of the isolates are inhibited, quinupristin-dalfopristin was 2 times more active against
S. aureus
isolates, 4 to 16 times more active against
S. haemolyticus
, and 8 to 32 times more active against
S. epidermidis
than vancomycin or teicoplanin.
Comparison of an Established Antibody Sandwich Method with an Inhibition Method of
Histoplasma capsulatum
Antigen Detection
Authors: Garringer Todd O. , Wheat L. Joseph , Brizendine Edward J.
Published: December 2019

The
Histoplasma
antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis. Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements. We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay. Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods. The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%,
P
= 0.500) and serum (82.5% versus 80.0%,
P
= 1.000). With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%,
P
= 1.000), and with urine, the specificity of the antibody sandwich method was superior (97.5% versus 80.0%,
P
= 0.039). While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = −0.0152, serum = 0.5595. Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470. Cross-reactivity was observed in specimens from patients infected with
Blastomyces dermatitidis
,
Paracoccidioides brasiliensis
, and
Penicillium marneffei
. In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range.
Sequence Analysis of the
ank
Gene of Granulocytic Ehrlichiae
Authors: Massung Robert F. , Owens Jessica H. , Ross David , Reed Kurt D. , Petrovec Miroslav , Bjoersdorff Anneli , Coughlin Richard T. , Beltz Gerald A. , Murphy Cheryl I.
Published: December 2019

The
ank
gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the
ank
gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota,
Ehrlichia equi
from California,
Ehrlichia phagocytophila
from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish
E. phagocytophila
. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the
E. equi
sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the
ank
gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.
Mapping of IS
6110
Insertion Sites in Two Epidemic Strains of
Mycobacterium tuberculosis
Authors: Beggs Marjorie L. , Eisenach Kathleen D. , Cave M. Donald
Published: December 2019

A widely distributed strain designated 210 was identified in a study of the diversity of
Mycobacterium tuberculosis
DNA fingerprints from three geographically separate states in the United States. This strain is characterized by a 21-band fingerprint pattern when probed with IS
6110
, and the pattern is similar to that displayed by strains designated W. Intracellular growth of strain 210 isolates in human macrophages is significantly faster than that of isolates from other clusters or nonclustered isolates. The purpose of this study was to identify the sites of IS
6110
insertions in strain 210 and compare these to IS
6110
insertion sites in strain W. Our hypothesis is that an IS
6110
insertion site(s) could possibly be responsible for a strain's increased capacity for transmission and/or replication. In this report, the insertion sites in strains 210 and W are described and referenced to their location in the
M. tuberculosis
H37Rv genome sequence. The W and 210 strains have 17 identical sites of IS
6110
insertion and additional sequence not found in H37Rv but present in other clinical isolates. The IS
6110
insertion site in the 36-bp direct repeat (DR) region of strains 210 and W has 15 spacers in the left flanking region. The DR region on the right side of IS
6110
has been deleted. Five sites of insertion in strain 210 not found in strain W are described, as well as two unique sites in strain W. One copy of IS
6110
was found to reside 55 bp in the
ctpD
gene. This gene is expressed, indicating that IS
6110
can provide a promoter sequence for the transcription of genes.
Molecular Analysis of H Antigens Reveals that Human Diarrheagenic
Escherichia coli
O26 Strains That Carry the
eae
Gene Belong to the H11 Clonal Complex
Authors: Zhang Wen-Lan , Bielaszewska Martina , Bockemühl Jochen , Schmidt Herbert , Scheutz Flemming , Karch Helge
Published: December 2019

Fifty-seven
Escherichia coli
O26 strains isolated from patients in six countries were investigated by PCR restriction fragment length polymorphism (RFLP) analysis of the flagellin-encoding (
fliC
) gene (
fliC
RFLP analysis). The strains were determined by serotyping to belong to five different H types or were nonmotile. The
fliC
RFLP analysis revealed only two different patterns among the 57 strains. One
fliC
RFLP pattern was displayed by 54 strains and was identical to that of
E. coli
H11 reference strain Su4321-41. The other
fliC
RFLP pattern was observed for three strains and was identical to that for
E. coli
H32 reference strain K10. The 54 strains with the H11
fliC
RFLP pattern included 22 strains of serotype O26:H11, 23 nonmotile strains, and 9 strains that were initially serotyped as H2, H8, H21, and H32 but that were confirmed to express H11 by repeat serotyping. All 54 strains with the H11
fliC
RFLP pattern contained the attaching-and-effacing (
eae
) gene. The three strains with the H32
fliC
RFLP pattern belonged to serotype O26:H32, and all were
eae
negative. The
fliC
genes of 14 selected
E. coli
O26:H11 strains isolated between 1964 and 1999 had identical nucleotide sequences. Our results demonstrate that
E. coli
O26 strains that carry the
eae
gene belong exclusively to the H11 clonal complex. Since there were no H11
fliC
allelic variations among the O26 strains tested,
E. coli
O26:H11 may have emerged recently. The
fliC
PCR-RFLP test is a reliable, easy-to-perform, and rapid method for determination of the H types of
E. coli
O26 isolates.
Clinical Significance and Antibiotic Resistance Patterns of
Leminorella
spp., an Emerging Nosocomial Pathogen
Authors: Blekher Lidia , Siegman-Igra Yardena , Schwartz David , Berger Stephen A. , Carmeli Yehuda
Published: December 2019

Although
Leminorella
spp., members of the family
Enterobacteriaceae
, were previously isolated from feces and urine specimens, clinical correlates have not been studied. We conducted a retrospective study to investigate the clinical significance and disease spectrum of these organisms, as well as their antibiotic susceptibility patterns. Identification and susceptibility testing were performed by an automated system. Eighteen cases were identified retrospectively during a 28-month period (1/97 to 4/99), representing an incidence of 11 cases per 100,000 patient admissions. The medical records of 14 patients were reviewed. The average patient age was 67 years, and 78% were males. Patients had multiple and diverse underlying conditions which might have predisposed them to infection.
Leminorella
spp. were classified as definite pathogens in 43% of the cases, probable pathogens in 29%, and possible pathogens in 21%. In one case of asymptomatic bacteriuria, the isolate had no clinical significance. All infections but one were nosocomial. Clinical syndromes included urinary tract infection in six patients, surgical site infection in three patients, and primary bacteremia, peritonitis, respiratory tract infection, and soft tissue infection in one patient each. Isolates were uniformly susceptible to imipenem. Other beta-lactam agents had poor activity against the isolates. We conclude that
Leminorella
spp. are significant nosocomial pathogens that are capable of causing a variety of clinical syndromes and are resistant to multiple antibiotic agents.
Urine
d
-Arabinitol/
l
-Arabinitol Ratio in Diagnosis of Invasive Candidiasis in Newborn Infants
Authors: Sigmundsdóttir Gudrún , Christensson Bertil , Björklund Lars J. , Håkansson Kristina , Pehrson Christina , Larsson Lennart
Published: December 2019

Infants treated in neonatal intensive care units suffer an increased risk for invasive candidiasis, but the diagnosis is sometimes difficult.
d
-arabinitol is a metabolite of most pathogenic
Candida
species. An elevated urine
d
-arabinitol/
l
-arabinitol (DA/LA) ratio is a sensitive sign of invasive candidiasis in children with cancer, but the method has not been previously evaluated for newborn infants. We therefore enrolled 117 infants in a neonatal intensive care unit, and 411 urine samples were obtained on filter paper. The DA/LA ratio was measured by gas chromatography-mass spectrometry. For 81 infants with no suspicion of superficial or invasive candidiasis, the urine DA/LA ratio was 2.7 ± 0.7 (mean ± standard deviation [SD]). The upper cutoff level was set at 4.8 (mean plus 3 SD). Of 22 infants with mucocutaneous candidiasis and not given systemic antifungal treatment, two had elevated DA/LA ratios, which normalized after removal of intravascular catheters. Eight other infants were given empiric antifungal treatment but had negative cultures; five of these had repeatedly elevated DA/LA ratios. Six infants with culture-positive invasive candidiasis all had one or more samples with elevated ratios. For seven infants, three with suspected and four with confirmed invasive candidiasis (for which follow-up samples were available), ratios normalized during antifungal treatment. In conclusion, urine DA/LA ratio determination is a rapid test and can be used for newborns. It is possibly more sensitive than fungal blood cultures in the diagnosis of invasive candidiasis and can also be used for monitoring the effect of antifungal treatment.
Molecular Identification of
Dialister pneumosintes
in Subgingival Plaque of Humans
Authors: Doan N. , Contreras A. , Flynn J. , Slots J. , Chen C.
Published: December 2019

Dialister pneumosintes
is a nonfermentative, anaerobic, gram-negative rod that grows with small, circular, transparent, shiny, smooth colonies on blood agar. Even though
D. pneumosintes
has been recovered from deep periodontal pockets, little is known about the relationship between the organism and destructive periodontal disease. This study describes a rapid PCR method to identify
D. pneumosintes
in periodontal samples. The PCR identification method detected as little as 10 pg of
D. pneumosintes
DNA or about 1 to 10 cells without nonspecific amplification of various periodontopathic bacteria. Twelve of 22 subgingival samples from adult periodontitis lesions yielded
D. pneumosintes
either by culture or by PCR identification. In culture-positive samples,
D. pneumosintes
averaged 3.9% (0.001 to 10.8%) of total isolates. Studies are needed to delineate virulence factors of
D. pneumosintes
pertinent to periodontal disease.