bluuebunny/crossref_metadata_2025 huggingface dataset:
Blinded, Externally Controlled Multicenter Evaluation of Light Microscopy and PCR for Detection of Microsporidia in Stool Specimens
Authors: Rinder Heinz , Janitschke Klaus , Aspöck Horst , Da Silva Alexandre J. , Deplazes Peter , Fedorko Daniel P. , Franzen Caspar , Futh Ursula , Hünger Frank , Lehmacher Anselm , Meyer Christian G. , Molina Jean-Michel , Sandfort Jörg , Weber Rainer , Löscher Thomas ,   the Diagnostic Multicenter Study Group on Microsporidia
Published: December 2019

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.
Detection of
Echinococcus multilocularis
in the Definitive Host: Coprodiagnosis by PCR as an Alternative to Necropsy
Authors: Dinkel Anke , von Nickisch-Rosenegk Markus , Bilger Birgit , Merli Michael , Lucius Richard , Romig Thomas
Published: December 2019

Recently, extensions of the range of
Echinococcus multilocularis
in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of
E. multilocularis
infections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the
E. multilocularis
mitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including
E. granulosus
. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with
E. multilocularis
amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites’ number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of
E. multilocularis
in carnivores.
Use of Monoclonal Antibodies To Identify Serotypes of Enterovirus Isolates
Authors: Rigonan Alma S. , Mann Linda , Chonmaitree Tasnee
Published: December 2019

Nonpoliovirus enteroviruses cause a variety of diseases that are common in young children and adults. The “gold standard” for laboratory diagnosis of enteroviruses is cell culture isolation, followed by serotype identification by neutralization assay. These procedures are time-consuming and expensive. Rapid serotype identification of enteroviruses is important in differentiating nonpoliovirus enterovirus pathogens from vaccine strain polioviruses that can be shed for some time after vaccination. In the present investigation, we evaluated a rapid method for serotype identification of enteroviruses by indirect immunofluorescence assay (IFA) using commercially available monoclonal antibodies for polioviruses, coxsackieviruses type B, and six serotypes of commonly circulating echoviruses. Of 291 isolates of enteroviruses included in the study, 95 were polioviruses and 196 were nonpoliovirus enteroviruses. Two hundred thirty-four of these (38 polioviruses and 196 nonpoliovirus enteroviruses) were consecutively grown in the laboratory over a 5-year period. IFA identified the serotypes of 74% of the consecutive isolates and 71% of all enterovirus isolates by yielding a positive staining result. The levels of agreement in the identification of the enterovirus group between IFA and neutralization tests were 92% for consecutively grown isolates and 85% for all enterovirus isolates. The sensitivity of the IFA for the detection of viruses for which specific monoclonal antibodies were applied was 73% for polioviruses, 85% for coxsackieviruses type B, and 94% for echoviruses. Specificity was near 100% for polioviruses and coxsackieviruses type B and 94% for echoviruses. We conclude that IFA can be helpful as a preliminary test for serotype identification of enteroviruses. The results are most accurate when the test identifies the isolate as a poliovirus.
Rapid Detection of
Chlamydia pneumoniae
by PCR-Enzyme Immunoassay
Authors: Jantos Christian A. , Roggendorf Rüdiger , Wuppermann Frederik N. , Hegemann Johannes H.
Published: December 2019

Chlamydia pneumoniae
is an important human respiratory pathogen. Laboratory diagnosis of infection with this organism is difficult. To facilitate the detection of
C. pneumoniae
by PCR, an enzyme immunoassay (EIA) for analysis of PCR products was developed. Biotin-labeled PCR products generated from the 16S rRNA gene of
C. pneumoniae
were hybridized to a digoxigenin-labeled probe and then immobilized to streptavidin-coated microtiter plates. Bound PCR product-probe hybrids were detected with antidigoxigenin peroxidase conjugate and a colorimetric substrate. This EIA was as sensitive as Southern blot hybridization for the detection of PCR products and 100 times more sensitive than visualization of PCR products on agarose gels. The diagnostic value of the PCR-EIA in comparison to cell culture was assessed in throat swab specimens from children with respiratory tract infections.
C. pneumoniae
was isolated from only 1 of 368 specimens tested. In contrast, 15 patient specimens were repeatedly positive for
C. pneumoniae
by PCR and Southern analysis. All of these 15 specimens were also identified by PCR-EIA. Of the 15 specimens positive by 16S rRNA-based PCR, 13 specimens could be confirmed by
omp1
-based PCR or direct fluorescent-antibody assay. Results of this study demonstrate that PCR is more sensitive than cell culture for the detection of
C. pneumoniae
. The EIA described here is a rapid, sensitive, and simple method for detection of amplified
C. pneumoniae
DNA.
Evaluation of the VITEK 2 System for Rapid Identification of Medically Relevant Gram-Negative Rods
Authors: Funke Guido , Monnet Dominique , deBernardis Chiara , von Graevenitz Alexander , Freney Jean
Published: December 2019

The new VITEK 2 system (bioMérieux) was evaluated at two independent sites with the identification card for gram-negative bacilli (ID-GNB card). Of the 845 strains tested, which represented 70 different taxa belonging to either the family
Enterobacteriaceae
or the nonenteric bacilli, 716 (84.7%) were correctly identified at the species level. Thirty-two (3.8%) additional strains were identified to the species level after the performance of simple, rapid manual tests (oxidase, hemolysis, indole reaction, motility, and pigmentation). For 80 (9.5%) strains, these additional tests did not lead to an identification at the species level but the correct species identification was given among the organisms listed. Only 7 (0.8%) strains were misidentified, and 10 (1.2%) were not identified. Mistakes were randomly distributed over different taxa. Due to the new, more sensitive fluorescence-based technology of the VITEK 2 system, final results were available after 3 h. Since our evaluation was mainly a stress test, it is predicted that the VITEK 2 system in conjunction with the ID-GNB card would perform well under conditions of a routine clinical laboratory in identifying members of the family
Enterobacteriaceae
and selected species of nonenteric bacteria. This system is a promising, highly automated new tool for the rapid identification of gram-negative bacilli from human clinical specimens.
Identification of a GranulocyticEhrlichiaStrain Isolated from a Horse in Switzerland and Comparison with Other Rickettsiae of theEhrlichia phagocytophilaGenogroup
Authors: Pusterla Nicola , Huder Jon B. , Feige Karsten , Lutz Hans
Published: December 2019

This case report describes a 12-year-old Arabian mare with granulocytic ehrlichiosis. Clinical signs included fever, apathy, anorexia, icterus, limb edema, and reluctance to move. Examination of buffy coat smears revealedEhrlichiaorganisms in neutrophils and eosinophils. A band of 1,428 bp was amplified from DNA of leukocytes via nested PCR and was identified as part of theEhrlichia16S rRNA gene. It differed from the gene sequences ofEhrlichia phagocytophilaandE. equiat two and three positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% identical to that of the agent of human granulocytic ehrlichiosis.
Distribution of
Staphylococcus
Species among Human Clinical Specimens and Emended Description of
Staphylococcus caprae
Authors: Kawamura Yoshiaki , Hou Xiao-Gang , Sultana Ferdousi , Hirose Kenji , Miyake Masaki , Shu Sin-Ei , Ezaki Takayuki
Published: December 2019

By DNA-DNA hybridization on microplates, we identified 1,230 strains of staphylococci from human clinical specimens and determined the distribution of species. The 10
Staphylococcus
species isolated most often were
S. epidermidis
(31.3%),
S. aureus
(23.3%),
S. haemolyticus
(12.2%),
S. caprae
(10.7%),
S. simulans
(4.4%),
S. hominis
(4.0%),
S. capitis
(3.9%),
S. saprophyticus
(3.6%),
S. warneri
(2.2%), and
S. lugdunensis
(1.3%). From these results, we realized that
S. caprae
strains were widely distributed in human clinical specimens. The description in
Bergey’s Manual of Systematic Bacteriology
indicates that no strains of
S. caprae
produce acid from fructose and mannitol, but all our
S. caprae
strains produced acid from fructose and mannitol. Consequently, many strains of
S. caprae
isolated from human clinical specimens have been misidentified as
S. haemolyticus
or
S. hominis
by conventional biochemical tests. In this paper, we propose an emended description of
S. caprae
.
Direct Susceptibility Testing with Positive BacT/Alert Blood Cultures by Using MicroScan Overnight and Rapid Panels
Authors: Waites Ken B. , Brookings E. S. , Moser S. A. , Zimmer B. L.
Published: December 2019

Studies were conducted on a method of direct inoculation of MicroScan dried overnight and of rapid panels with positive aerobic blood cultures obtained from the BacT/Alert to determine antimicrobial susceptibilities. Inocula were limited to specimens that appeared unimicrobic on Gram stain. Results were compared to those obtained from panels inoculated following subculture. For 133 gram-negative bacilli, there were 94.7 and 93.5% categorical agreements between direct and standard methods for all drugs tested with overnight and rapid panels, respectively. For 104 gram-positive cocci, there were 93.2 and 93.1% categorical agreements for overnight and rapid panels, respectively. The major error (false resistance) rate for gram negatives was 1.4% for overnight versus 0.7% for rapid panels. The very major error (false susceptibility) rate was 2.7% for overnight versus 8.1% for rapid panels. The total error rates were 1.6% for overnight panels and 1.5% for rapid panels. The major error rates for gram-positive direct susceptibility tests were 2.6% for overnight and 2.5% for rapid panels. The very major error rates were 8.8 and 7.2% for overnight and rapid panels, respectively. Total error rates were 3.6% for overnight and rapid gram-positive panels. These findings suggest that susceptibility results obtained from directly inoculated gram-negative overnight panels have the greatest correlation to those obtained by standard methods. When discrepant results occur with direct-susceptibility testing, they are more likely to show false susceptibility than false resistance.
Simple, Inexpensive, Reliable Method for Differentiation of
Candida dubliniensis
from
Candida albicans
Authors: Pinjon Emmanuelle , Sullivan Derek , Salkin Ira , Shanley Diarmuid , Coleman David
Published: December 2019

Candida dubliniensis
is a recently described pathogenic species which shares many phenotypic features with
Candida albicans
, including the ability to form germ tubes and chlamydospores. These similarities have caused significant problems in the identification of
C. dubliniensis
by the average clinical mycology laboratory. To facilitate the differentiation of these species, we investigated the growth of 120 isolates of
C. dubliniensis
and 98
C. albicans
isolates at 42 and 45°C on Emmons’ modified Sabouraud glucose agar (SGA) and 10 isolates of each species in yeast-peptone-dextrose broth. None of the
C. dubliniensis
isolates grew on the agar or in the broth medium at 45°C, while 11 isolates were capable of growing on SGA at 42°C. In contrast, all of the
C. albicans
isolates but one grew at 45°C on or in either medium. These reproducible results clearly demonstrate that the incubation of isolates suspected to be
C. dubliniensis
or
C. albicans
at 45°C provides a simple, reliable, and inexpensive method for the differentiation of the two species.
Evidence of Toscana Virus Variants Circulating in Tuscany, Italy, during the Summers of 1995 to 1997
Authors: Valassina Marcello , Cuppone Anna Maria , Bianchi Silvia , Santini Laura , Cusi Maria Grazia
Published: December 2019

The Toscana virus can cause neurological infection in adults. This study of 112 cases of acute meningitis which occurred during the summers of 1995, 1996, and 1997 demonstrated the presence of viral RNA in the cerebrospinal fluid of 56 patients. Their sequence analysis shows four variants of the Toscana virus.
Replication of Bovine Herpesvirus Type 4 in Human Cells In Vitro
Authors: Egyed László
Published: December 2019

A reference strain (Movár 33/63) of bovine herpesvirus type 4 (BHV-4) was inoculated into 14 different human cell lines and five primary cell cultures representing various human tissues. BHV-4 replicated in two embryonic lung cell lines, MRC-5 and Wistar-38, and in a giant-cell glioblastoma cell culture. Cytopathic effect and intranuclear inclusion bodies were observed in these cells. PCR detected a 10,000-times-higher level of BHV-4 DNA. Titration of the supernatant indicated a 100-fold increase of infectious particles. Since this is the first bovine (human herpesvirus 8 and Epstein-Barr virus related) herpesvirus which replicates on human cells in vitro, the danger of possible human BHV-4 infection should not be ignored.
Diagnosis of Kala-Azar by Nested PCR Based on Amplification of the
Leishmania
Mini-Exon Gene
Authors: Katakura Ken , Kawazu Shin-Ichiro , Naya Toshimitsu , Nagakura Koichi , Ito Mamoru , Aikawa Masamichi , Qu Jing-Qi , Guan Li-Ren , Zuo Xin-Pin , Chai Jun-Jie , Chang K.-P. , Matsumoto Yoshitsugu
Published: December 2019

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the
Leishmania
genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single
Leishmania
parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.
Evaluation of the Biostar Chlamydia OIA Assay with Specimens from Women Attending a Sexually Transmitted Disease Clinic
Authors: Pate Mitchell S. , Dixon Paula B. , Hardy Kim , Crosby Mark , Hook E. W.
Published: December 2019

Chlamydia trachomatis
infections are the most prevalent sexually transmitted diseases (STDs) in the United States. In acute-care settings such as clinics and emergency rooms, a desirable chlamydia screening assay should exhibit good sensitivity and good specificity and should provide test results while the patient is still present. The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) that provides test results in less than 30 min and that uses a test format that allows office-based testing. This assay is performed entirely at room temperature without the need for rotators or other specialized equipment. The goal of this study was to compare the performance of the Biostar Chlamydia OIA for the detection of
C. trachomatis
with the performance of cell culture, direct fluorescent-antibody (DFA) assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), and PCR (Roche Amplicor
Chlamydia trachomatis
; Roche, Branchburg, N.J.) for the detection of
C. trachomatis
infections in women attending an urban STD clinic. For calculations of relative test performance (sensitivity, specificity, and positive and negative predictive values), patient specimens that yielded positive results by two or more of the four assays (cell culture, DFA assay, PCR, and OIA) were classified as “true infections.” By these criteria, 42 of 306 total specimens were classified as positive for
C. trachomatis
(positive prevalence, 13.7%), 11 (3.6%; 10 by PCR and 1 by DFA assay) were positive by a single assay, and 253 (82.7%) were negative by all four tests. All culture-positive specimens were also positive by at least one other assay. Among the culture-negative specimens, 14 (5%) specimens were positive by two of the three non-culture-based assays used. By using the criterion that positivity by at least two of the tests indicated a true infection, the relative sensitivities were as follows: culture and PCR, 92.9% each; Biostar Chlamydia OIA, 73.8%; and DFA assay, 59.5%.
Emergence of Vancomycin-Resistant Enterococci in Australia: Phenotypic and Genotypic Characteristics of Isolates
Authors: Bell Jan M. , Paton James C. , Turnidge John
Published: December 2019

Enterococci with resistance to glycopeptides have recently emerged in Australia. We developed multiplex PCR assays for
vanA
,
vanB
,
vanC1
, and
vanC2
or
vanC3
in order to examine the genetic basis for vancomycin resistance in Australian isolates of vancomycin-resistant
Enterococcus faecium
and
E. faecalis
(VRE). The predominant genotype from human clinical
E. faecium
isolates was
vanB
. The PCR
van
genotype was consistent with the resistance phenotype in all but six cases. One
vanA E. faecalis
isolate had a VanB phenotype, one
vanB E. faecium
isolate had a VanA phenotype, and four
E. faecalis
isolates were consistently negative for
vanA
,
vanB
,
vanC1
, and
vanC2
or
vanC3
, even though they exhibited a VanB phenotype. These four isolates were subsequently examined for the presence of
vanD
by published methods and were found to be negative. No vancomycin-susceptible strains produced a PCR product. On the basis of our findings the epidemiology of VRE in Australia appears to be different from that in either the United States or Europe. Our multiplex PCR assays gave a rapid and accurate method for determining the genotype and confirming the identification of glycopeptide-resistant enterococci. Rapid and accurate methods are essential, because laboratory-based surveillance is critical in programs for the detection, control, and prevention of the transmission of glycopeptide-resistant enterococci.
Direct Identification of
Coxiella burnetii
Plasmids in Human Sera by Nested PCR
Authors: Zhang G. Q. , Hotta A. , Mizutani M. , Ho T. , Yamaguchi T. , Fukushi H. , Hirai K.
Published: December 2019

Nested PCR assays were used for the direct identification of
Coxiella burnetii
plasmids in human sera. A total of 81 serum samples from 81 patients with Q fever were tested by nested PCR with four sets of primers. The first set of primers was used to detect the genomic sequences. The second set of primers was used to detect the conserved sequences of the plasmids. Another two sets of primers were used to identify the QpH1 and QpRS plasmids. QpH1 and QpRS plasmid-specific sequences were identified in 40 (49.4%) and 24 (29.6%) of the serum samples, respectively. Both of the QpH1 and QpRS plasmid-specific sequences were detected in 5 (8.6%) of the serum samples but were not found in 12 (20.7%) of the serum samples. Furthermore, all of the 23 acute-phase serum samples were positive for the QpH1 plasmid and negative for the QpRS plasmid. Nested PCR with plasmid-specific primers appears to be a useful method for the direct typing of
C. burnetii
plasmids in human sera.
Variants of the 3′ Region of the
cagA
Gene in
Helicobacter pylori
Isolates from Patients with Different
H. pylori
-Associated Diseases
Authors: Yamaoka Yoshio , Kodama Tadashi , Kashima Kei , Graham David Y. , Sepulveda Antonia R.
Published: December 2019

The CagA protein of
Helicobacter pylori
is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3′ region of the
cagA
gene contains repeated sequences. To determine whether there are structural changes in the 3′ region of
cagA
that predict outcome of
H. pylori
infection, we examined 155
cagA
gene-positive
H. pylori
isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3′ region of the
cagA
gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3′ region of the
cagA
gene in Japan differs markedly from the primary sequence of
cagA
genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (
P
< 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in
cagA
genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among
cagA
-positive
H. pylori
strains.
Detection and Differentiation of
vanC-1
,
vanC-2
, and
vanC-3
Glycopeptide Resistance Genes in Enterococci
Authors: Clark Nancye C. , Teixeira Lucia M. , Facklam Richard R. , Tenover Fred C.
Published: December 2019

The VanC phenotype, as found in
Enterococcus gallinarum
,
E. casseliflavus
, and
E. flavescens
, is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the
vanC-1
gene in
E. gallinarum
, the
vanC-2
gene in
E. casseliflavus
, and the
vanC-3
gene in
E. flavescens
have been reported, although there is some disagreement as to whether
E. flavescens
is a legitimate enterococcal species. Previous attempts to differentiate the
vanC-2
and
vanC-3
genes by PCR analysis have been unsuccessful. The purpose of the present study was to detect and differentiate the three
vanC
determinants and examine the distribution of these genes in a collection of both typical and atypical enterococci. The 796-bp
vanC-1
PCR product was amplified only from
E. gallinarum
isolates. As expected, due to the extensive homology in the
vanC-2
and
vanC-3
gene sequences, all of the
E. casseliflavus
and
E. casseliflavus/flavescens
isolates produced the 484-bp
vanC-2
PCR product, although the
E. gallinarum
isolates were negative. Only the
E. casseliflavus/flavescens
isolates produced the 224-bp
vanC-3
product. Using the three sets of primers, we were able to detect and distinguish the
vanC-1
,
vanC-2
, and
vanC-3
genes from both typical and atypical enterococci strains. Antimicrobial susceptibility tests and analysis of genomic DNA by pulsed-field gel electrophoresis were also performed, but the results indicated that they were not able to distinguish among strains possessing the three
vanC
genotypes.
Isolation ofPorphyromonas gingivalisand Detection of Immunoglobulin A Specific to Fimbrial Antigen in Gingival Crevicular Fluid
Authors: Condorelli Francesca , Scalia Guido , Calì Giuditta , Rossetti Bruno , Nicoletti Giuseppe , Lo Bue Anna M.
Published: December 2019

The present study evaluated the prevalence ofPorphyromonas gingivalisand the correlation between the bacterial culture method and the detection of immunoglobulin A (IgA) specific to theP. gingivalisfimbrial antigen in gingival crevicular fluid (GCF).P. gingivaliswas isolated from 78.3% of subgingival plaque samples obtained from active sites and 34.7% of those from inactive sites of periodontal patients.P. gingivaliswas isolated from only 4.7% of healthy subjects (control group). Immunoglobulins specific to theP. gingivalisfimbrial antigen were detected by enzyme-linked immunosorbent assay (ELISA). The overall agreement between the results of theP. gingivalisculture method and the results of specific IgA detection in periodontal patients was 71.7% for active sites and 58.7% for inactive sites. IgA specific toP. gingivaliswas absent in GCF from all of the sites of healthy subjects. The results suggest thatP. gingivalisis associated with the local production of specific IgA. The detection of IgA antibodies specific toP. gingivalisin GCF by ELISA may be used as a predictive parameter to reveal the early phase of the activation of recurrent periodontal infections.
Identification of a Rough Strain of
Escherichia coli
O157:H7 That Produces No Detectable O157 Antigen
Authors: Feng Peter , Sandlin Robin C. , Park Choong H. , Wilson Richard A. , Nishibuchi Mitsuaki
Published: December 2019

MA6, an O157:H7-like strain, did not react with most anti-O157 kits examined; however, it had the
rfbE
gene that is essential for O157 expression and carried O157:H7 virulence factors. Lipopolysaccharide analysis showed that MA6 is a rough strain that does not produce the O157 antigen, but genetically, it belongs in the O157:H7 clonal group.
Inhibition of PCR in Genital and Urine Specimens Submitted for
Chlamydia trachomatis
Testing
Authors: Toye B. , Woods W. , Bobrowska M. , Ramotar K.
Published: December 2019

We determined the frequency of PCR inhibition in genital and urine specimens submitted for
Chlamydia trachomatis
testing using the internal control DNA provided with the COBAS AMPLICOR
C. trachomatis
test and assessed methods to remove it. Inhibition occurred in 65 of 906 (7%) cervical swabs, 23 of 51 (45%) urethral swabs, and 2 of 175 (1.1%) urine samples. Overall, inhibition was eliminated in processed specimens after storage at 4°C in 77 of 90 specimens (86%), freezing at −70°C in 59 of 82 specimens (72%), storage at 4°C followed by either 1:100 dilution in 37 of 43 specimens (86%) or 1:10 dilution in 42 of 47 specimens (89%), and phenol-chloroform extraction in 79 of 80 specimens (99%). No positive specimens were missed due to inhibition. We conclude that PCR inhibition is rare with urine specimens and infrequent with endocervical swabs but occurs frequently with urethral swabs. The frequency of PCR inhibition may be significantly reduced by methods which can be easily incorporated into the processing of specimens.
Purification of
Encephalitozoon
Cultures Contaminated by Mycoplasmas by Murine Intraperitoneal Inoculation
Authors: Ridoux Olivier , Foucault Cédric , Drancourt Michel
Published: December 2019

Encephalitozoon
species are strict intracellular microsporidia. Cocultures with eukaryotic cell lines can become accidently contaminated by mycoplasmas. We propose a decontamination protocol based on differential cell targeting after intraperitoneal inoculation in mice. Mycoplasma-free microsporidia were isolated from the brains and spleens of inoculated mice 24 h postinoculation by using the centrifugation shell vial system. Identification was confirmed by direct sequencing of PCR-amplified 16S rRNA.
Colony Size Can Be Used To Determine the MIC of Fluconazole for Pathogenic Yeasts
Authors: Xu Jianping , Vilgalys Rytas , Mitchell Thomas G.
Published: December 2019

This report describes a new statistical method for estimating the MIC of fluconazole for yeasts pathogenic to humans. This method is based on comparison of the colony sizes on solid media containing different concentrations of fluconazole. By this method, the MICs of fluconazole for 10 yeast strains were comparable to results obtained by the standard method recommended by the National Committee for Clinical Laboratory Standards. This method is simple to perform and easy to interpret. The turnaround time is faster than other methods. The method should be applicable to the determination MICs of other antifungal drugs for yeasts.
Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland
Authors: Gorgievski-Hrisoho Meri , Schumacher Jean-Daniel , Vilimonovic Nevenka , Germann Daniel , Matter Lukas
Published: December 2019

Enteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions.
Cooperative Oligonucleotides Mediating Direct Capture of Hepatitis C Virus RNA from Serum
Authors: O’Meara Deirdre , Yun Zhibing , Sönnerborg Anders , Lundeberg Joakim
Published: December 2019

A novel method for direct capture of hepatitis C virus (HCV) RNA from clinical samples has been developed. This approach takes advantage of the cooperative interactions between adjacently hybridized oligonucleotides. Here, this cooperative effect was combined with solid-phase technology, whereby a capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. When these contiguously hybridized probes were used for the extraction of HCV RNA from clinical samples, the capture efficiency was increased up to 25-fold in comparison to capture with a single probe. The applicability of this sample preparation assay was further investigated by performing a comparative study with both a conventional guanidinium extraction method and a commercial quantitative assay.
Necessity of Molecular Techniques To Distinguish between
Neisseria meningitidis
Strains Isolated from Patients with Meningococcal Disease and from Their Healthy Contacts
Authors: Vogel Ulrich , Morelli Giovanna , Zurth Kerstin , Claus Heike , Kriener Eugen , Achtman Mark , Frosch Matthias
Published: December 2019

Serogroup C strains of
Neisseria meningitidis
were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient’s nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.
Determination of Copy Number of rRNA Genes in
Pneumocystis carinii
f. sp.
hominis
Authors: Tang Xing , Bartlett Marilyn S. , Smith James W. , Lu Jang-Jih , Lee Chao-Hung
Published: December 2019

Differential PCR was performed to determine the copy number of rRNA genes in
Pneumocystis carinii
f. sp.
hominis
. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with
P. carinii
pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that
P. carinii
f. sp.
hominis
has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of
P. carinii
f. sp.
hominis
.
Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire
Authors: Nkengasong John N. , Kalou Mirielle , Maurice Chantal , Bile Celestin , Borget Marie-Yolande , Koblavi Stéphania , Boateng Emmanuel , Sassan-Morokro Madeleine , Anatole-Ehounou Ekpini , Ghys Peter , Greenberg Alan E. , Wiktor Stefan Z.
Published: December 2019

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (
n
= 67 [96%]; mean RNA levels, 5.2 log
10
HIV-1 RNA copies/ml) than the NucliSens assay (
n
= 56 [80%]; 4.3 log
10
HIV-1 RNA copies/ml) or the standard HIV Monitor assay (
n
= 44 [63%]; mean RNA levels, 3.8 log
10
HIV-1 RNA copies/ml) (all
P
values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (
r
= 0.76;
P
< 0.001) and the standard HIV Monitor assay (
r
= 0.57;
P
< 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (
r
= 0.60;
P
< 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (
r
= −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.
Antigenic Characterization of Hantaan and Seoul Virus Nucleocapsid Proteins Expressed by Recombinant Baculovirus: Application of a Truncated Protein, Lacking an Antigenic Region Common to the Two Viruses, as a Serotyping Antigen
Authors: Morii Mayuko , Yoshimatsu Kumiko , Arikawa Jiro , Zhou Guizen , Kariwa Hiroaki , Takashima Ikuo
Published: December 2019

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus
Hantavirus
in the family
Bunyaviridae
and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.
Expanding Allelic Diversity of
Helicobacter pylori vacA
Authors: van Doorn Leen-Jan , Figueiredo Céu , Sanna Ricardo , Pena Salvador , Midolo Peter , Ng Enders K. W. , Atherton John C. , Blaser Martin J. , Quint Wim G. V.
Published: December 2019

The diversity of the gene encoding the vacuolating cytotoxin (
vacA
) of
Helicobacter pylori
was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of
vacA
, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from
H. pylori
isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different
vacA
s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (
cagA
). This PCR-LiPA method permits rapid analysis of the
vacA
and
cagA
status of
H. pylori
strains for clinical and epidemiological studies and will facilitate identification of any further variations.
Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis
Authors: van Vliet K. E. , Glimåker M. , Lebon P. , Klapper P. E. , Taylor C. E. , Ciardi M. , van der Avoort H. G. A. M. , Diepersloot R. J. A. , Kurtz J. , Peeters M. F. , Cleator G. M. , van Loon A. M.
Published: December 2019

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (
P
< 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.
Diagnostic Value of PCR for Detection ofBorrelia burgdorferiin Skin Biopsy and Urine Samples from Patients with Skin Borreliosis
Authors: Brettschneider S. , Bruckbauer H. , Klugbauer N. , Hofmann H.
Published: December 2019

Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined forBorrelia burgdorferiDNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients.B. burgdorferiwas cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. ElevatedB. burgdorferiantibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology.
Use of Laboratory Assays To Predict Cytomegalovirus Disease in Renal Transplant Recipients
Authors: Tong Cheuk Yan William , Cuevas Luis , Williams Helen , Bakran Ali
Published: December 2019

Eight laboratory assays, viz., the pp65 direct antigenemia test, a quantitative cytomegalovirus (CMV)-specific immunoglobulin G (IgG) assay (Biomerieux VIDAS), a CMV-specific IgM assay (Biomerieux VIDAS), the Hybrid Capture system (Murex), an in-house PCR with plasma (P-PCR) and leukocytes (L-PCR), and a commercial PCR (Roche AMPLICOR) with plasma (P-AMP) and leukocytes (L-AMP), were compared for their abilities to predict CMV disease before the onset of illness in a prospective study of 37 renal transplant recipients. By using an expanded criterion for active infection (two or more of the markers positive) and a clinical definition of disease, 22 (59%) patients were identified as having active CMV infection and 13 (35%) were identified as having CMV disease. Of the 13 CMV-seronegative recipients who received seropositive kidneys (R− group), 8 had active infection and disease. All assays were 100% specific and 100% predictive of CMV disease in the R− group. The leukocyte PCRs (L-PCR and L-AMP) were the most sensitive assays, had positive results an average of between 8 and 13 days before the onset of illness, and were the assays of choice. The performance of the assays was less satisfactory for the 24 patients who were CMV seropositive before transplantation (R+ group). A negative result was more useful for this group. Overall, P-AMP had the best results, and it could be the assay of choice for monitoring R+ patients. The non-PCR-based methods generally had high specificities but often gave late positive results and were not sensitive enough for use as prediction tools for either group of patients.