bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated Quantitative Analysis of Hepatitis B Virus DNA by Using the Cobas Amplicor HBV Monitor Test
Authors: Noborg Ulrika , Gusdal Annkatrin , Pisa Eva K. , Hedrum Anders , Lindh Magnus
Published: December 2019

A highly sensitive method of quantitative analysis of hepatitis B virus (HBV) DNA in serum, the Cobas Amplicor HBV Monitor (Cobas-AM) test, was evaluated. Following a manual extraction of viral DNA, amplification, colorimetric detection, and quantitative determination are all automatically performed in the Cobas analyzer. Serially diluted samples with known HBV DNA concentrations were analyzed blindly. All samples with a virus concentration of 400 copies/ml and 83% of samples with a virus concentration of 100 copies/ml could be detected. A linear correlation between input HBV DNA and measured HBV DNA was seen in the range from 100 to 10
5
copies/ml. The mean coefficient of variation was 29.6% for all input levels and 18.9% for HBV DNA concentrations above 400 copies/ml. Samples with an HBV DNA level above 10
9
copies/ml could be reproducibly measured after predilution to 10
−4
or 10
−6
in negative serum; however, the level was underestimated if target DNA after dilution was still above the linear range of the assay. Quantitative results of the Cobas-AM test were interchangeable with measurements by the manual microwell plate version of Amplicor HBV Monitor (MWP-AM); the mean ratio for log Cobas-AM results/log MWP-AM results was 0.97 (standard error of the mean, 0.007) when serum samples from 153 chronic carriers were analyzed. The test should be of value for clinical assessment of chronic carriers and for monitoring the response to antiviral treatment. A limitation is the relatively narrow linear range of the assay, requiring predilution of high-titer (mainly hepatitis B e-antigen-positive) samples.
Use of Molecular Assays in Diagnosis and Monitoring of Cytomegalovirus Disease following Renal Transplantation
Authors: Aitken Celia , Barrett-Muir Winsome , Millar Colin , Templeton Kate , Thomas Janice , Sheridan Fran , Jeffries Donald , Yaqoob Magdi , Breuer Judith
Published: December 2019

We compared two commercial molecular assays (the Murex Hybrid Capture CMV DNA assay [HCA], version 2, and the Roche Amplicor plasma PCR assay) with a standard shell vial assay in detecting and predicting cytomegalovirus (CMV) disease in a group of renal transplant patients and assessed the role of viral load measurements (using the HCA) in their management. The sensitivity of the HCA and Amplicor assay in terms of disease detection was 100%, compared to 71% for the shell vial assay. Both the HCA and the PCR assay detected all cases of disease, at medians of 11 and 12.5 days before the onset of symptoms, respectively. Significantly higher viral loads were detected in those patients with symptoms (7.9 × 10
5
copies/ml) than in patients without symptoms (7.9 × 10
4
copies/ml;
P
< 0.0001). There was also a trend towards higher viral loads in those patients with primary infections (7.8 × 10
5
copies/ml) than in those patients with reactivations of CMV disease or reinfections. Successful treatment with ganciclovir was associated with a >90% reduction in viral load. Both of these new assays are sensitive and easy to use. A comparison of accurate quantitation is also useful in monitoring responses to antiviral therapy.
Community Strain of Methicillin-Resistant
Staphylococcus aureus
Involved in a Hospital Outbreak
Authors: O’Brien F. G. , Pearman J. W. , Gracey M. , Riley T. V. , Grubb W. B.
Published: December 2019

Western Australia (WA) has been able to prevent methicillin-resistant
Staphylococcus aureus
(MRSA) strains from outside of the state from becoming established in its hospitals. Recently, a single-strain outbreak of MRSA occurred in a WA metropolitan teaching hospital following admission of an infected patient from a remote community. The strain responsible for the outbreak was unrelated to any imported strains and spread rapidly in the hospital. Screening of two remote communities in the region from which the index case came revealed that 42% of the people in one community and 24% in the other carried MRSA. Isolates were typed by resistance pattern, plasmid analysis, contour-clamped homogeneous electric field electrophoresis, bacteriophage pattern, and coagulase gene restriction fragment length polymorphism. It was found that of the people carrying MRSA, 39% in the former community and 17% in the latter community were carrying an MRSA strain which was indistinguishable from the strain that caused the hospital outbreak.
Antigenic Domains of the Open Reading Frame 2-Encoded Protein of Hepatitis E Virus
Authors: Khudyakov Yury E. , Lopareva Elena N. , Jue Danny L. , Crews Tamara K. , Thyagarajan S. P. , Fields Howard A.
Published: December 2019

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.
Genotypes of Canine Distemper Virus Determined by Analysis of the Hemagglutinin Genes of Recent Isolates from Dogs in Japan
Authors: Mochizuki Masami , Hashimoto Michiru , Hagiwara Shigeyuki , Yoshida Yoshio , Ishiguro Shinryo
Published: December 2019

Canine distemper of domestic dogs is caused by canine distemper virus (CDV), a member of the morbilliviruses. It has been a highly contagious disease of great veterinary importance for centuries, but for the last several decades it has been controlled satisfactorily by modified live vaccines. In the 1990s, however, it was described that CDV strains genetically different from vaccine strains may have caused the disease in vaccinated dogs. The highest antigenic variation is found in the H protein. Therefore, in the present study, hemagglutinin (H) genes obtained from current vaccines and field isolates and amplified directly from clinical specimens were genetically analyzed by restriction fragment length polymorphism assay and sequencing. Phylogenetic analysis of the H-gene amino acid sequences revealed that at least two CDV genotypes are circulating among dogs in Japan; one is a genotype to which almost all Japanese CDV isolates belong and the other has not been previously described. Both are separate and independent from the other lineages or genotypes of vaccine strains, as well as European and U.S. CDV isolates. The results suggest that CDV has also evolved in Japan, and further studies will be needed for an evaluation and possible improvement of the efficacies of current CDV vaccines.
Hepatitis B Virus (HBV) Mutations Associated with Resistance to Lamivudine in Patients Coinfected with HBV and Human Immunodeficiency Virus
Authors: Thibault Vincent , Benhamou Yves , Seguret Christophe , Bochet Marie , Katlama Christine , Bricaire François , Opolon Pierre , Poynard Thierry , Agut Henri
Published: December 2019

Mutations associated with hepatitis B virus (HBV) resistance to lamivudine have not been extensively addressed in human immunodeficiency virus (HIV)-HBV coinfection. We have studied the HBV polymerase sequences from nine coinfected patients who experienced HBV recurrence while under lamivudine treatment. In seven of these nine patients, Met
550
, belonging to the highly conserved YMDD motif, was mutated to Val and was associated with a substitution of Met for Leu
526
in each case. In the two remaining patients, we found a Met
550
-to-Ile change that was associated in only one case with a Leu
526
-to-Met mutation. No mutation was observed in three control patients not receiving lamivudine. This study demonstrates the emergence of particular genetic profiles in HBV-HIV-coinfected patients experiencing a loss of control of HBV infection despite high doses of lamivudine.
Phenotypic and Genetic Characterization of a Novel
Borrelia burgdorferi
Sensu Lato Isolate from a Patient with Lyme Borreliosis
Authors: Wang Guiqing , van Dam Alje P. , Dankert Jacob
Published: December 2019

Borrelia burgdorferi
sensu lato A14S was cultured from a skin biopsy specimen of a patient with erythema migrans in The Netherlands. This isolate had a unique DNA fingerprint pattern compared to 135 other
B. burgdorferi
sensu lato isolates. In this study, the isolate A14S was further characterized by protein analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reactivity with various monoclonal antibodies. In addition, the 16S rRNA,
ospA
, and
ospC
genes, as well as the 5S-23S rRNA intergenic spacer DNA, were amplified by PCR, cloned, and sequenced. SDS-PAGE protein profiles and phylogenetic analysis based on all of the analyzed genes confirmed that
B. burgdorferi
sensu lato A14S was phenotypically and genetically different from the three human pathogenic species
B. burgdorferi
sensu stricto,
Borrelia garinii
, and
Borrelia afzelii
, as well as from other
B. burgdorferi
sensu lato species. Our findings indicate that
Borrelia
genomic groups or isolates other than the three well-known human pathogenic species may also cause human Lyme borreliosis.
Rapid Detection of Methicillin Resistance in
Staphylococcus aureus
Isolates by the MRSA-Screen Latex Agglutination Test
Authors: van Leeuwen Willem B. , van Pelt Cindy , Luijendijk Ad , Verbrugh Henri A. , Goessens Wil H. F.
Published: December 2019

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the
mecA
PCR (“gold standard”) for the detection of methicillin resistance in
Staphylococcus aureus
. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant
S. aureus
(MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the
mecA
gene by exposure to methicillin. Both
mecA
PCR and MRSA-Screen displayed negative results among the methicillin-susceptible
S. aureus
strains (
n
= 106), as well as for
Micrococcus
spp. (
n
= 10), members of the family
Enterobacteriaceae
(
n
= 10),
Streptococcus pneumoniae
(
n
= 10), and
Enterococcus
spp. (
n
= 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant
Staphylococcus epidermidis
strains score positived in both the latex test and the
mecA
PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory.
Analysis by Enzyme-Linked Immunosorbent Assay and PCR of Human Liver Abscess Aspirates from Patients in China for
Entamoeba histolytica
Authors: Zengzhu G. , Bracha R. , Nuchamowitz Y. , Cheng-I W. , Mirelman D.
Published: December 2019

Aspirates of liver abscesses were analyzed for
Entamoeba histolytica
. PCR detected a gene encoding a 30-kDa protein in all samples but detected the ribosomal DNA gene in only 14 (33.3%) samples. Enzyme-linked immunosorbent assay detected antigen in 41 (97.6%) samples. PCR analysis of a strain-specific antigen (SSG) revealed that abscesses were caused by various strains.
Evaluation of the Effects of Storage in Two Different Swab Fabrics and under Three Different Transport Conditions on Recovery of Aerobic and Anaerobic Bacteria
Authors: Roelofsen E. , van Leeuwen M. , Meijer-Severs G. J. , Wilkinson M. H. F. , Degener J. E.
Published: December 2019

Recovery of six anaerobic and five aerobic pathogens from viscose swabs and polyurethane swabs (Culturette EZ) was evaluated quantitatively, and transport in aerobic dry tubes, aerobic Amies transport medium (Transwab), and anaerobic universal transport medium (Port-a-Cul) was compared. The Culturette EZ in aerobic dry tubes gave the highest recovery levels. Data obtained with clinical specimens confirmed these results.
Evaluation of the Leukocyte Esterase and Nitrite Urine Dipstick Screening Tests for Detection of Bacteriuria in Women with Suspected Uncomplicated Urinary Tract Infections
Authors: Semeniuk Heather , Church Deirdre
Published: December 2019

A positive dipstick urinalysis (i.e., leukocyte esterase test and/or nitrite test) did not reliably detect significant bacteriuria in 479 ambulatory women with suspected uncomplicated urinary tract infection; 18.9% of the urine samples that demonstrated significant bacteriuria would have been rejected by the laboratory based on a negative urinalysis screen.
Characterization of Enterovirus Isolates from Patients with Heart Muscle Disease in a Selenium-Deficient Area of China
Authors: Peng Tianqing , Li Yanwen , Yang Yingzhen , Niu Cunlong , Morgan-Capner Peter , Archard Leonard C. , Zhang Hongyi
Published: December 2019

An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural areas of southwestern China has been established. Enteroviruses have been isolated from patients with KD or during outbreaks of myocarditis in last two decades. Six of these isolates grew readily in cell lines (Vero or HEp-2) and were investigated by a novel molecular typing method apart from serotyping and pathogenicity. A neutralization assay identified two isolates from KD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isolates, also from myocarditis. Direct nucleotide sequencing of reverse transcription-PCR products amplified from the 5′ nontranslated region (5′NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisting of coxsackie B-like viruses, including some echovirus serotypes. Sequence analysis of the coding region for viral capsid protein VP1 showed that two isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknown serotypes, are most closely related to the sequence of CVB6. Sequences among these isolates varied from 82.3 to 99% in the 5′NTR and from 69 to 99% in VP1, indicating no cross contamination. The pathogenicity of these viruses in adult and suckling mice was assessed. None caused pathologic changes in the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominant in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogenicity.
Rapid Differentiation of Fermentative from Nonfermentative Gram-Negative Bacilli in Positive Blood Cultures by an Impedance Method
Authors: Chang Tsung Chain , Huang Ay Huey
Published: December 2019

Rapid differentiation of fermentative gram-negative bacilli (fermenters) from nonfermentative gram-negative bacilli (nonfermenters) in positive blood cultures may help physicians to narrow the choice of appropriate antibiotics for empiric treatment. An impedance method for direct differentiation of fermenters from nonfermenters was investigated. The bacterial suspensions (or positive culture broths containing gram-negative bacteria) were inoculated into the module wells of a Bactometer (bioMérieux, Inc., Hazelwood, Mo.) containing 1 ml of Muller-Hinton broth. The inoculated modules were incubated at 35°C, and the change in impedance in each well was continuously monitored. The amount of time required to cause a series of significant deviations from baseline impedance values was defined as the detection time (DT). The percent change of impedance was defined as the change of impedance at the time interval from DT to DT plus 1 h. After testing 857 strains of pure cultures (586 strains of fermenters and 271 strains of nonfermenters), a breakpoint (2.98%) of impedance change was obtained by discriminant analysis. Strains displaying impedance changes of greater than 2.98% were classified as fermenters; the others were classified as nonfermenters. By using this breakpoint, 98.6% (340 of 345) of positive blood cultures containing fermenters and 98% (98 of 100) of positive blood cultures containing nonfermenters were correctly classified. The impedance method was simple, and the results were normally available within 2 to 4 h after direct inoculation of positive blood culture broths.
Evaluation of
d
-Xylose and 1% Methyl-α-
d
-Glucopyranoside Fermentation Tests for Distinguishing
Enterococcus gallinarum
from
Enterococcus faecium
Authors: Chen D. K. , Pearce L. , McGeer A. , Low D. E. , Willey B. M.
Published: December 2019

To determine the validity of the rapid xylose and methyl-α-
d
-glucopyranoside (MDG) fermentation tests in distinguishing
Enterococcus gallinarum
from
Enterococcus faecium
, 156 well-characterized clinical isolates of enterococci (55
E. gallinarum
, 91
E. faecium
, and 10
Enterococcus faecalis
isolates) known to be of different clones were examined in a blinded fashion. Species identification was confirmed by PCR of the
ddl
ligase genes of
E. faecium
and
E. faecalis
and the
vanC1
gene of
E. gallinarum
. Xylose tests were performed with
d
-xylose tablets by using a heavy bacterial suspension and were interpreted after 2 h of incubation. Standard MDG fermentation tests were read after 24 h of incubation. The xylose fermentation test had a sensitivity of 98% (54 of 55) and a specificity of 99% (100 of 101) in distinguishing
E. gallinarum
from
E. faecium
and
E. faecalis
. The standard MDG test had a sensitivity of 100% (55 of 55) and a specificity of 95% (96 of 101) after 24 h. The xylose fermentation test is a simple method, easily incorporated into laboratory protocols, that distinguishes
E. gallinarum
from
E. faecium
with high sensitivity and specificity in 2 h. The standard MDG test has high sensitivity and can be useful in ruling out the presence of
E. gallinarum
but requires overnight incubation.
Genotyping of Rotaviruses in Environmental Water and Stool Samples in Southern Switzerland by Nucleotide Sequence Analysis of 189 Base Pairs at the 5′ End of the VP7 Gene
Authors: Baggi Franca , Peduzzi Raffaele
Published: December 2019

Stool specimens from children (<4 years old) with diarrhea were collected over a 1-year period in Ticino (southern region of Switzerland). During the same period, environmental samples were collected from surface waters in the proximity of major water treatment plants. From treatment plants, samples were collected from the raw sewage and before the release of the treated water. From rivers, samples were collected before and after receiving the treated waters. A single-step reverse transcription (RT)-PCR amplification of the entire VP7 gene from extracted double-stranded RNA was developed. For the water samples, a further nested PCR was necessary to increase sensitivity. All amplified viral products were sequenced, and the sequence profile was compared to that of the VP7 genes of human and animal rotaviruses from GenBank. Rotavirus strains are characterized by outer capsid proteins G (glycoprotein) and P (protease-cleaved protein). Correct G genotyping of viral sequences from stool and water samples was possible by analyzing only 189 bp at the 5′ end of the VP7 gene. In the Ticino region, the most predominant G genotype among clinical and water samples was G1. Genotypes G2 and G4 were found only among clinical samples. We also detected rotavirus G1-type sequences in feces from a healthy adult. This finding corroborates the hypothesis that healthy adults act as potential reservoirs for the spread of rotavirus in the environment. In our experiments, this RT-PCR-based method for rotavirus genotyping has proven to be a useful tool for epidemiological investigations.
Immunodiagnosis of
Echinococcus
Infections: Confirmatory Testing and Species Differentiation by a New Commercial Western Blot
Authors: Liance Martine , Janin Veronique , Bresson-Hadni Solange , Vuitton Dominique-Angele , Houin Rene , Piarroux Renaud
Published: December 2019

The
Echinococcus
Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from
Echinococcus multilocularis
, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to
Echinococcus granulosus
, and alveolar echinococcosis, due to
E. multilocularis
. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of
Echinococcus
-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between
E. granulosus
- and
E. multilocularis
-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available
Echinococcus
Western Blot IgG for the serological confirmation of human echinococcosis.
Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens
Authors: Martin Cara , Roberts David , van der Weide Marjo , Rossau Rudi , Jannes Geert , Smith Terry , Maher Majella
Published: December 2019

We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including
Candida
,
Aspergillus
, and
Cryptococcus
species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of
Candida albicans
,
Candida parapsilosis
,
Candida glabrata
,
Candida tropicalis
,
Candida krusei
,
Candida dubliniensis
,
Cryptococcus neoformans
,
Aspergillus fumigatus
,
Aspergillus versicolor
,
Aspergillus nidulans
and
Aspergillus flavus
. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from
C. albicans
,
A. fumigatus
, and
C. neoformans
. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with
Candida
cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.
Comparison of the CMV Brite Turbo Assay and the Digene Hybrid Capture CMV DNA (Version 2.0) Assay for Quantitation of Cytomegalovirus in Renal Transplant Recipients
Authors: Ho Stephen K. N. , Li Fu-Keung , Lai Kar-Neng , Chan Tak-Mao
Published: December 2019

We compared the CMV Brite Turbo Kit (BT) and the Digene Hybrid Capture CMV DNA (version 2.0) assay (HC2) in the quantitation of pp65 antigenemia and cytomegalovirus (CMV) DNA levels in immunosuppressed renal transplant recipients. Of 123 blood specimens collected from 24 renal transplant recipients, BT and HC2 assays detected 35 and 39 positive samples, respectively. The overall concordance rate between the two assays was 90%. Discordant results were observed at low levels of viremia, so that 8 samples were HC2 positive but BT negative and another 4 were BT positive but HC2 negative. There was good correlation (
R
2
= 0.766;
P
< 0.01) between the levels of CMV DNA and pp65 antigenemia in the 31 concordant positive samples. Correlation between results obtained with the two assays was confirmed by longitudinal studies for a patient who developed clinical CMV disease. HC2 may be more sensitive at low viremia levels and allow earlier detection of impending CMV disease. The BT assay offered the advantage of a rapid (2-h) turnaround time. We conclude that BT and HC2 assays have similar sensitivity and efficacy in the diagnosis and monitoring of CMV infection and disease in renal transplant recipients. While the HC2 assay would be appropriate for centers that handle a large number of samples, the BT test may be more suitable for small sample numbers or when results are needed urgently.
Molecular Epidemiology of
Entamoeba
spp.: Evidence of a Bottleneck (Demographic Sweep) and Transcontinental Spread of Diploid Parasites
Authors: Ghosh Sudip , Frisardi Marta , Ramirez-Avila Lynn , Descoteaux Steven , Sturm-Ramirez Katherine , Newton-Sanchez Oscar Alberto , Santos-Preciado Jose Ignacio , Ganguly Chaiti , Lohia Anuradha , Reed Sharon , Samuelson John
Published: December 2019

Entamoeba histolytica
causes amebic colitis and liver abscess in developing countries such as Mexico and India.
Entamoeba dispar
is morphologically identical but is not associated with disease. Here we determined the ploidy of
E. histolytica
and developed PCR-based methods for distinguishing field isolates of
E. histolytica
or
E. dispar
. Fluorescence in situ hybridization showed that
E. histolytica
trophozoites are diploid for five “single-copy” probes tested. Intergenic sequences between
superoxide dismutase
and
actin 3
genes of clinical isolates of
E. histolytica
from the New and Old Worlds were identical, as were those of
E. dispar
. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast,
E. histolytica
and
E. dispar
genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic.
chitinase
alleles suggested that the early axenized strains of
E. histolytica
, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar
E. dispar
parasites can be identified in both the New and Old Worlds.
Ser-rich protein
alleles, which suggested the presence of the HM-1 strain in Mexico City, included some
E. histolytica
genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at
chitinase
and
Ser-rich protein
loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of
E. histolytica
and
E. dispar
.
Evaluation of the Abbott LCx HIV-1 RNA Quantitative, a New Assay for Quantitative Determination of Human Immunodeficiency Virus Type 1 RNA
Authors: Zanchetta Nadia , Nardi Giampiero , Tocalli Loredana , Drago Lorenzo , Bossi Carla , Pulvirenti Francesco Renato , Galli Claudio , Gismondo Maria Rita
Published: December 2019

A new quantitative reverse transcription (RT)-PCR assay for human immunodeficiency virus type 1 (HIV-1) RNA (Abbott LCx HIV RNA Quantitative assay) has been compared with the Organon NucliSens assay on 521 retrospective samples obtained from HIV-1-positive patients monitored during highly active antiretroviral therapy, 79 of whom were assayed also by the Chiron Quantiplex 3.0 system and on characterized panels. The LCx system showed a moderate correlation (
r
= 0.795) and gave higher results than the NucliSens system on 245 of 327 concordant positive samples, with similar sensitivity. Correlation with Quantiplex system results was higher (
r
= 0.943). LCx reproducibility was very good; the procedure was simple, well controlled, and rapid (up to 48 results in 7 h). The HIV RNA quantitative assay on the LCx system is suitable for routine use.
Results of a Quality Assurance Program for Detection of Cytomegalovirus Infection in the Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted Human Immunodeficiency Virus Infection Study
Authors: Demmler Gail J. , Istas Allison , Easley Kirk A. , Kovacs Andrea ,   the Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted HIV-1 Infection Study Group
Published: December 2019

A quality assurance program was established by the Pediatric Pulmonary and Cardiovascular Complications of Vertically Transmitted Human Immunodeficiency Virus Type 1 Infection Study Group for monitoring cytomegalovirus (CMV) antibody and culture results obtained from nine different participating laboratories. Over a 3-year period, every 6 months, each laboratory was sent by the designated reference laboratory six coded samples: three urine samples for CMV detection and three serum samples for CMV immunoglobulin G (IgG) and IgM antibody determination. Overall, the participating laboratories exhibited the following composite performance statistics, relative to the reference laboratory (sensitivity and specificity, respectively): 100 and 97.4% for CMV cultures, 95.5 and 94.4% for CMV IgG antibody assays, and 92.6 and 90.2% for CMV IgM assays. The practice of having individual laboratories use different commercial methods and reagents for CMV detection and antibody determination was successfully monitored and provided useful information on the comparable performance of different assays.
Diversity of Domain V of 23S rRNA Gene Sequence in Different
Enterococcus
Species
Authors: Tsiodras Sotirios , Gold Howard S. , Coakley Eoin P. G. , Wennersten Christine , Moellering Robert C. , Eliopoulos George M.
Published: December 2019

The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628;
Escherichia coli
numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of
Enterococcus
was amplified by PCR. Twenty enterococcal isolates were tested, including
Enterococcus faecium
(six isolates),
Enterococcus faecalis
,
Enterococcus avium
,
Enterococcus durans
,
Enterococcus gallinarum
,
Enterococcus casseliflavus
(two isolates of each), and
Enterococcus raffinosus
,
Enterococcus mundtii
,
Enterococcus malodoratus
, and
Enterococcus hirae
(one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from
E. faecium
and
E. faecalis
differed from those of all other enterococci. The domain V sequences of
E. durans
and
E. hirae
were identical. This was also true for
E. gallinarum
and
E. casseliflavus. E. avium
differed from
E. casseliflavus
by 23 bases, from
E. durans
by 16 bases, and from
E. malodoratus
by 2 bases.
E. avium
differed from
E. raffinosus
by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.
Molecular and Pathogenic Characterization ofBorrelia burgdorferiSensu Lato Isolates from Spain
Authors: Escudero Raquel , Barral Marta , Pérez Azucena , Vitutia M. Mar , García-Pérez Ana L. , Jiménez Santos , Sellek Ricela E. , Anda Pedro
Published: December 2019

FifteenBorrelia burgdorferisensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nineBorrelia garinii, including the two human isolates, threeB. burgdorferisensu stricto, twoB. valaisiana, and oneB. lusitaniae), showing a diverse spectrum ofB. burgdorferisensu lato species.B. gariniiisolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. SevenB. gariniiisolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the twoB. valaisianaisolates also caused disease in mice. Only oneB. burgdorferisensu stricto isolate was recovered from the urinary bladder. One isolate each ofB. valaisianaandB. lusitaniaewere not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity ofB. burgdorferisensu lato in areas in northern Spain where Lyme disease is endemic.
Real-Time Quantitative PCR for Human Herpesvirus 6 DNA
Authors: Locatelli Giuseppe , Santoro Fabio , Veglia Fabrizio , Gobbi Alberto , Lusso Paolo , Malnati Mauro S.
Published: December 2019

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5′ nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10
6
viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.
Coyotes (Canis latrans) as the Reservoir for a Human PathogenicBartonellasp.: Molecular Epidemiology ofBartonella vinsoniisubsp.berkhoffiiInfection in Coyotes from Central Coastal California
Authors: Chang Chao-Chin , Kasten Rickie W. , Chomel Bruno B. , Simpson Darren C. , Hew Carrie M. , Kordick Dorsey L. , Heller Remy , Piemont Yves , Breitschwerdt Edward B.
Published: December 2019

Bartonella vinsoniisubsp.berkhoffiiwas originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible withBartonellainfection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic withB. vinsoniisubsp.berkhoffiiand 83 animals (76%) hadB. vinsoniisubsp.berkhoffiiantibodies. These findings suggest these animals could be the wildlife reservoir ofB. vinsoniisubsp.berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of thegltAand 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those ofB. vinsoniisubsp.berkhoffii. Partial sequencing of thegltAand 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate andB. vinsoniisubsp.berkhoffii(ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31Bartonellabacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsoniisubsp.berkhoffiiATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) withSmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, threeBartonellabacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission ofB. vinsoniisubsp.berkhoffii, especially to identify potential vectors, and to determine how humans become infected.
Serodiagnosis of
Bartonella bacilliformis
Infection by Indirect Fluorescence Antibody Assay: Test Development and Application to a Population in an Area of Bartonellosis Endemicity
Authors: Chamberlin Judith , Laughlin Larry , Gordon Scott , Romero Sofia , Solórzano Nelson , Regnery Russell L.
Published: December 2019

Bartonella bacilliformis
causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to
B. bacilliformis
and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting
B. bacilliformis
antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where
B. bacilliformis
is endemic and where the point prevalence of infection is 45%.
Avian Pneumovirus (APV) RNA from Wild and Sentinel Birds in the United States Has Genetic Homology with RNA from APV Isolates from Domestic Turkeys
Authors: Shin Hyun-Jin , Njenga M. Kariuki , McComb Brian , Halvorson David A. , Nagaraja Kakambi V.
Published: December 2019

Nasal turbinates or swabs were collected from wild ducks, geese, owls, sparrows, swallows, and starlings and from sentinel ducks placed next to turkey farms experiencing avian pneumovirus (APV) infections and were analyzed for APV genome and infectious particles. APV RNA was detected in samples examined from geese, sparrows, and starlings. APV RNA and antibodies were also detected in two different groups of sentinel ducks. Infectious APV was recovered from sentinel duck samples. The APV M gene isolated from the wild birds had over 96% predicted amino acid identity with APV/Minnesota 2A, which was isolated earlier from domestic turkeys showing respiratory illness, suggesting that wild birds may be involved in spreading APV infection.
Application of Representational Difference Analysis to Genomic Fragments of Marek's Disease Virus
Authors: Endoh Daiji , Cho Kyoung-Oh , Tsukamoto Kenji , Morimura Toshifumi , Kon Yasuhiro , Hayashi Masanobu
Published: December 2019

A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from
Bam
HI-,
Bgl
II-,
Eco
RI-,
Hin
dIII-, or
Xho
I-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct sequencing of the MDV genome. We detected an interstrain difference in the size of restriction enzyme-digested fragments on agarose gel. This method was used on a single feather pulp to generate sufficient MDV DNA for cloning.
Rapid Genotyping of Varicella-Zoster Virus Vaccine and Wild-Type Strains with Fluorophore-Labeled Hybridization Probes
Authors: Loparev Vladimir N. , McCaustland Karen , Holloway Brian P. , Krause Philip R. , Takayama Michiko , Schmid D. Scott
Published: December 2019

We developed a single-tube rapid method for the detection and differentiation of varicella-zoster virus (VZV) vaccine and wild-type strains that combines rapid-cycle PCR with wild-type-specific fluorescent probe melting profiles for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescence-labeled hybridization probes. During the annealing step of the thermal cycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manipulation. In addition to reducing the time required to produce a result, this method substantially reduces the risk of contamination of the final product as well as the risk of sample tracking errors. The genotypes of 79 VZV-positive samples determined by this fluorescent resonance energy transfer (FRET) method were identical to the genotypes obtained by conventional PCR and restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable for typing and that is also practical for use for the processing of large numbers of specimens.
Dissemination of CTX-M-3 and CMY-2 β-Lactamases among Clinical Isolates of
Escherichia coli
in Southern Taiwan
Authors: Yan Jing-Jou , Ko Wen-Chien , Tsai Shu-Huei , Wu Hsiu-Mei , Jin Ying-Tai , Wu Jiunn-Jong
Published: December 2019

A total of 1,210 clinical isolates of
Escherichia coli
collected from a university hospital in southern Taiwan were screened for production of extended-spectrum β-lactamases (ESBLs). Expression of classical ESBLs (resistant to extended-spectrum β-lactam agents and susceptible to β-lactam inhibitors) was inferred in 18 isolates by the phenotypic confirmatory test. These included 10 isolates producing CTX-M-3, 2 strains carrying SHV-12, 1 strain harboring SHV-5, 1 strain expressing TEM-10, and 4 strains producing unidentifiable ESBLs with a pI of 8.05, 8.0, or 7.4. Eighteen isolates that showed decreased susceptibilities to ceftazidime and/or cefotaxime, negative results for the confirmatory test, and high-level resistance to cefoxitin (MICs of ≥128 μg/ml) were also investigated. Five isolates were found to produce CMY-2 AmpC enzymes, one isolate carried both CTX-M-3 and CMY-2, and the remaining three and nine isolates expressed putative AmpC β-lactamases with pIs of >9.0 and 8.9, respectively. Thus, together with the isolate producing CTX-M-3 and CMY-2, 19 (1.6%) isolates produced classical ESBLs. Pulsed-field gel electrophoresis revealed that all isolates carrying CTX-M-3 and/or CMY-2 were genetically unrelated, indicating that dissemination of resistance plasmids was responsible for the spread of these two enzymes among
E. coli
in this area. Among the 16 isolates expressing CTX-M-3 and/or CMY-2, 5 might have colonized outside the hospital environment. Our data indicate that CTX-M-3 and CMY-2, two β-lactamases initially identified in Europe, have been disseminated to and are prevalent in Taiwan.
Comparison of Sequencing of the
por
Gene and Typing of the
opa
Gene for Discrimination of
Neisseria gonorrhoeae
Strains from Sexual Contacts
Authors: Viscidi Raphael P. , Demma James C. , Gu Jing , Zenilman Jonathan
Published: December 2019

Typing of gonococcal strains is a valuable tool for the biological confirmation of sexual contacts. We have developed a typing method based on DNA sequencing of two overlapping
por
gene fragments generated by a heminested PCR. We compared sequencing of the
por
gene (POR sequencing) and typing of the
opa
gene (OPA typing) for the characterization of strains from 17 sexual partnerships. Both methods were highly discriminatory. A different genotype was detected in 15 of the 17 epidemiologically unconnected couples by POR sequencing and in 16 of the 17 couples by OPA typing with restriction enzyme
Hpa
II. Within partnerships, identical genotypes were obtained from 16 of the 17 known sex contacts by POR sequencing and from 15 of the 17 by OPA typing. Compared to OPA typing, which relies on interpretation of bands in a gel, DNA sequence data offer the advantage of being objective and portable. As costs for sequencing decline, the method should become affordable for most laboratory personnel who wish to type gonococcal strains.