bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Group C Rotaviruses Associated with Diarrhea Outbreaks in Feeder Pigs
Authors: Kim Yunjeong , Chang Kyeong-Ok , Straw Barbara , Saif Linda J.
Published: December 2019

Feces and serum specimens were collected from three farms in Michigan on which ∼50-lb (8- to 9-week-old) pigs experienced diarrhea just after placement into all-in-all-out finishing barns. The clinical signs (profuse watery diarrhea lasting about 2 weeks and no vomiting) were similar on all farms, and the morbidity rate was high (ranging from 60 to 80%) but without mortality. Eleven diarrheic fecal samples from the farms were tested for group A and C rotaviruses by immune electron microscopy (IEM) and various assays. IEM indicated that the fecal samples reacted only with antiserum against group C rotaviruses, and polyacrylamide gel electrophoresis indicated that the samples had characteristic genomic electropherotypes for group C rotavirus. Group C rotavirus was detected by cell culture immunofluorescence (CCIF) tests in nine fecal samples, but no group A rotavirus was detected by enzyme-linked immunosorbent assay or CCIF. By reverse transcription (RT)-PCR, all 11 fecal samples were positive for group C rotaviruses, with only 2 samples positive for group A rotaviruses. However, a second amplification of RT-PCR products using nested primers detected group A rotaviruses in all samples. Analysis of nucleotide and deduced amino acid sequences of the RT-PCR product (partial-length VP7) of the group C rotavirus showed 87.2 to 91% nucleotide identity and 92.6 to 95.9% amino acid identity among two strong samples from the different farms and the Cowden strain of porcine group C rotavirus. All nine convalescent-phase serum samples tested had neutralizing antibodies to the Cowden strain, and the majority of them had neutralizing antibody against group A rotaviruses (OSU or/and Gottfried strains) by fluorescent focus neutralization tests. Although group C rotaviruses have been reported as a cause of sporadic diarrhea in suckling or weanling pigs, to our knowledge, this is the first report of epidemic diarrhea outbreaks associated with group C rotavirus in older pigs.
A Highly Sensitive Assay for Detection and Quantitation of Human Cytomegalovirus DNA in Serum and Plasma by PCR and Electrochemiluminescence
Authors: Boom René , Sol Cees , Weel Jan , Gerrits Yvette , de Boer Monique , Wertheim-van Dillen Pauline
Published: December 2019

We describe a diagnostic PCR assay (D-PCR) and a quantitative PCR assay (Q-PCR) for the detection of human cytomegalovirus (CMV) in plasma and serum. In the D-PCR, DNA was purified from plasma or serum together with internal control (IC) DNA, which monitored both DNA extraction efficiency and PCR efficiency. DNA was subjected to PCR with a single primer pair, and the amount of PCR products was determined by electrochemiluminescence (ECL) in the QPCR System 5000 (Perkin-Elmer) after hybridization with Tris (2,2′-bipyridine) ruthenium (II) chelate-labeled probes. The lower limit of sensitivity of the D-PCR was reached at about 25 CMV particles/ml. Even with extremely low DNA inputs (four molecules of IC DNA/200 μl of plasma), very high yields (near 100%) were reached. DNA extracted from specimens that were CMV positive by the D-PCR was subsequently used in the Q-PCR, which was similar to the D-PCR. The viral load was calculated directly from the ratio of CMV and IC signals obtained by ECL. The Q-PCR assay is quantitative in the range of 100 to 150,000 copies of CMV/ml, independent of the anticoagulant. Interassay variation, intra-assay variation, and interspecimen variation were about 25%, suggesting that the Q-PCR will reliably detect fourfold differences in viral load. Comparison of paired serum and plasma specimens from CMV-infected individuals showed that serum CMV loads were frequently more than 10-fold lower than plasma CMV loads.
New Enzyme Immunoassays for Sensitive Detection of Circulating
Candida albicans
Mannan and Antimannan Antibodies: Useful Combined Test for Diagnosis of Systemic Candidiasis
Authors: Sendid Boualem , Tabouret Marc , Poirot Jean Louis , Mathieu Daniel , Fruit Jeanine , Poulain Daniel
Published: December 2019

Two standardized enzyme immunoassays for the serological diagnosis of candidiasis were developed. The first one detects antimannan antibodies, while the second one detects mannan with a sensitivity of 0.1 ng/ml. These tests were applied to 162 serum samples retrospectively selected from 43 patients with mycologically and clinically proven candidiasis caused by
Candida albicans
. Forty-three serum samples were positive for mannan, and 63 had significant antibody levels. Strikingly, only five serum samples were simultaneously positive by both tests. When the results were analyzed per patient, 36 (84%) presented at least one serum positive by one test. For 30 of them, positivity by one test was always associated with negative results by the other test for any of the tested sera. For six patients whose sera were positive for either an antigen or an antibody response, a balance between positivity by each test was evidenced by kinetic analysis of sera drawn during the time course of the infection. Controls consisted of 98 serum samples from healthy individuals, 93 serum samples from patients hospitalized in intensive care units, and 39 serum samples from patients with deep mycoses. The sensitivities and specificities were 40 and 98% and 53 and 94% for mannanemia or antibody detection, respectively. These values reached 80 and 93%, respectively, when the results of both tests were combined. These observations, which clearly demonstrate a disparity between circulation of a given mannan catabolite and antimannan antibody response, suggest that use of both enzyme immunoassays may be useful for the routine diagnosis of candidiasis.
Rapid PCR Test for Discriminating between
Candida albicans
and
Candida dubliniensis
Isolates Using Primers Derived from the pH-Regulated
PHR1
and
PHR2
Genes of
C. albicans
Authors: Kurzai Oliver , Heinz Werner J. , Sullivan Derek J. , Coleman David C. , Frosch Matthias , Mühlschlegel Fritz A.
Published: December 2019

The development of a satisfactory means to reliably distinguish between the two closely related species
Candida albicans
and
Candida dubliniensis
in the clinical mycology laboratory has proved difficult because these two species are phenotypically so similar. In this study, we have detected homologues of the pH-regulated
C. albicans PHR1
and
PHR2
genes in
C. dubliniensis
. Restriction fragment length polymorphism analysis suggests that there are significant sequence differences between the genes of the two species. In order to exploit this apparent difference, oligonucleotide primers based on the coding sequence of the
C. albicans PHR1
structural gene were designed and used in PCR experiments. Use of these primers with
C. albicans
template DNA from 17 strains yielded a predicted 1.6-kb product, while
C. dubliniensis
template DNA from 19 strains yielded no product. We therefore propose that PCR using these primers is a rapid and reliable means of distinguishing the two germ tube- and chlamydospore-producing species
C. albicans
and
C. dubliniensis
.
Evaluation of MRSA-Screen, a Simple Anti-PBP 2a Slide Latex Agglutination Kit, for Rapid Detection of Methicillin Resistance in
Staphylococcus aureus
Authors: Cavassini Matthias , Wenger Aline , Jaton Katia , Blanc Dominique S. , Bille Jacques
Published: December 2019

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the
mecA
gene for the detection of methicillin resistance in
Staphylococcus aureus
. A total of 120 methicillin-susceptible
S. aureus
(MSSA) and 80 methicillin-resistant
S. aureus
(MRSA) isolates, defined by the absence or presence of the
mecA
gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.
Use of Nucleic Acid Probes for Identification of
Mycobacterium tuberculosis
Directly from MB/BacT Bottles
Authors: Badak F. Zuhre , Goksel Servet , Sertoz Ruchan , Nafile Bedii , Ermertcan Safak , Cavusoglu Cengiz , Bilgic Altinay
Published: December 2019

The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study. A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants. The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT. Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe. Centrifugation of the contents of the bottle was not done prior to probe testing. The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of
M. tuberculosis
and 32 isolates of mycobacteria other than
M. tuberculosis
[MOTT]). A total of 96.4% (213 of 221) of the bottles growing
M. tuberculosis
produced a positive direct DNA probe result for
M. tuberculosis
complex. One hundred percent (16 of 16) of the bottles growing
M. gordonae
produced a positive direct DNA probe result for
M. gordonae
. A total of 3.6% (8 of 221) of the bottles growing
M. tuberculosis
did not yield a positive direct DNA probe result for
M. tuberculosis
complex. The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight
M. tuberculosis
isolates. Two (0.9%)
M. tuberculosis
isolates gave a negative result for the
M. tuberculosis
probe test applied on the MB/BacT broth and its subculture. The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for
M. gordonae
,
M. avium
,
M. intracellulare
, and
M. kansasii
probes. The sensitivity and specificity of AccuProbe for the identification of
M. tuberculosis
and
M. gordonae
directly from MB/BacT broth were 96.4 and 100% for
M. tuberculosis
and 100 and 100% for
M. gordonae
, respectively. The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of
M. tuberculosis
and
M. gordonae
.
Disk Diffusion Method for Determining Susceptibilities of
Candida
spp. to MK-0991
Authors: Lozano-Chiu Mario , Nelson Page W. , Paetznick Victor L. , Rex John H.
Published: December 2019

We have developed an agar-based methodology for testing susceptibilities of
Candida
spp. to the new antifungal agent MK-0991, a glucan synthase inhibitor. Results obtained with this method correlated well with the results obtained by the National Committee for Clinical Laboratory Standards M27-A broth microdilution reference method. However, as noted with prior comparisons of broth- and agar-based systems, some isolates yielded inhibition zones which were not consistent with the MICs obtained for them. Understanding the implications of these differences will require testing in an in vivo system.
High Prevalence of GB Virus C in Brazil and Molecular Evidence for Intrafamilial Transmission
Authors: Pinho João R. R. , Zanotto Paolo M. De A. , Ferreira João L. P. , Sumita Laura M. , Carrilho Flair J. , da Silva Luiz C. , Capacci M. Lourdes , Silva Adávio O. , Guz Betty , Gonçales Fernando L. , Gonçales Neiva S. L. , Buck Gregory A. , Meyers Gregory A. , Bernardini A. Plínio
Published: December 2019

The prevalence of GB virus C (GBV-C) in candidate Brazilian blood donors with normal and elevated alanine aminotransferase levels was found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected with HCV. Molecular characterization of GBV-C by partial sequencing of the NS3 region showed clustering between members of a single family, implying intrafamilial transmission. In conclusion, these results together suggest that contagion mechanisms which facilitate intrafamilial transmission of GBV-C may partially explain the high prevalence of viremic carriers worldwide.
Incidence of Upper Respiratory Tract
Mycoplasma pneumoniae
Infections among Outpatients in Rhône-Alpes, France, during Five Successive Winter Periods
Authors: Layani-Milon Marie-Paule , Gras Isabelle , Valette Martine , Luciani Jacques , Stagnara Jean , Aymard Michèle , Lina Bruno
Published: December 2019

In this prospective study, nasal swab samples from patients with acute respiratory infections were evaluated for the presence of
Mycoplasma pneumoniae
. This PCR-plus-hybridization-based detection was associated with the detection of other viral agents. During the five winter surveillance periods, 3,897 samples were collected by 75 medical practitioners participating in the Groupe Régional d’Observation de la Grippe surveillance network in Rhône-Alpes (France).
M. pneumoniae
was detected in 283 samples (7.3%); its rate of detection ranged from 10.1 to 2.0% over the five periods, and it was the second most frequently isolated pathogen during the survey, after influenza A. Three high-prevalence winters were observed, yielding an early winter peak of
M. pneumoniae
infection which was observed in all age groups. No statistically significant difference was detected between rates of infections in the different age groups, but
M. pneumoniae
infection was significantly related to lower respiratory tract infection during periods of high prevalence. This study defined the frequency of
M. pneumoniae
detection from nasal swab specimens in patients with acute respiratory infections, confirming its high prevalence and the presence of large outbreaks due to this pathogen.
Epidemiological Study of Paratuberculosis in Wild Rabbits in Scotland
Authors: Greig Alastair , Stevenson Karen , Henderson Dennis , Perez Valentin , Hughes Valerie , Pavlik Ivo , Hines Murray E. , McKendrick Iain , Sharp J. Michael
Published: December 2019

A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease.
The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms
Authors: Ceri H. , Olson M. E. , Stremick C. , Read R. R. , Morck D. , Buret A.
Published: December 2019

Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains:
Escherichia coli
ATCC 25922,
Pseudomonas aeruginosa
ATCC 27853, and
Staphylococcus aureus
ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (
P
> 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.
Design and Evaluation of a Human Immunodeficiency Virus Type 1 RNA Assay Using Nucleic Acid Sequence-Based Amplification Technology Able To Quantify Both Group M and O Viruses by Using the Long Terminal Repeat as Target
Authors: de Baar Michel P. , van der Schoot Audrey M. , Goudsmit Jaap , Jacobs Femke , Ehren Ron , van der Horn Karin H. M. , Oudshoorn Peter , de Wolf Frank , de Ronde Anthony
Published: December 2019

Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used
gag
-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than are
gag
-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide.
Susceptibility Testing of Anaerobic Bacteria: Evaluation of the Redesigned (Version 96) bioMérieux ATB ANA Device
Authors: Dubreuil L. , Houcke I. , Singer E.
Published: December 2019

We compared the susceptibility results for 200 clinical anaerobes with nine antibiotics obtained by using a new ATB ANA (bioMérieux) device against those obtained by the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method. For better evaluation of the device, we added some resistant
Bacteroides fragilis
group strains from our own collection: 3, 6, and 12 strains that were resistant to imipenem, ticarcillin plus clavulanic acid, and co-amoxiclav, respectively, and 2 other strains with decreased susceptibility to metronidazole. For some strains that did not grow on ATB S medium, tests were performed by using West-Wilkins medium supplemented with 1.5% agar. The new ATB ANA device made clinical categorization of the investigated strains possible, according to French (Committee of the Antibiogram of the French Society of Microbiology) or U.S. (NCCLS) breakpoints, with the following respective results: category agreement, 94.3 and 94.9%; minor errors, 4.8 and 3.8%; major errors, 0.4 and 0.8%; and very major errors 4.6 and 4.2%. The ATB ANA device was able to detect low-level metronidazole-resistant
B. fragilis
strains according to the French breakpoints but not the NCCLS ones. For
B. fragilis
and β-lactamase-positive
Prevotella
strains, the clustering effect of amoxicillin MICs around the French breakpoints led to more frequent minor errors. ATB ANA is a very convenient method to determine the antibiotic susceptibilities of anaerobes. Results obtained by ATB ANA correlated well with those obtained by the reference method.
Nasopharyngeal Carriage of Penicillin-Resistant
Streptococcus pneumoniae
among Children with Acute Respiratory Tract Infections in Thailand: a Molecular Epidemiological Survey
Authors: Dejsirilert Surang , Overweg Karin , Sluijter Marcel , Saengsuk Leelawadee , Gratten Mike , Ezaki Takayuki , Hermans Peter W. M.
Published: December 2019

The prevalence of penicillin-resistant
Streptococcus pneumoniae
in Thailand has dramatically increased over the last decade. During a national survey, which was conducted from 1992 to 1994, 37.2% of the pneumococci isolated from the nasopharynges of children with acute respiratory tract infections were penicillin resistant (MIC, ≥0.1 μg/ml). In order to investigate the prevalence and clonal relatedness of nasopharyngeal carriage of penicillin-resistant
S. pneumoniae
in Thailand, a molecular epidemiological survey was undertaken. To this end, 53 penicillin-resistant pneumococcal isolates from children who suffered from acute respiratory tract infections and who originated from five distinct regions of the country were characterized in detail. DNA fingerprint analysis demonstrated 13 clusters, i.e., genotypes shared by two or more strains, and 14 unique genotypes. The cluster size varied from 2 (nine clusters) to 11 strains (one cluster). Six of the 13 restriction fragment end labeling clusters consisted of two or more distinct serotypes, indicating frequent horizontal transfer of capsular genes. Geographical distribution of the genotypes among the five regions of Thailand demonstrated that only four genetic clusters were restricted to single areas of the country, whereas the other nine clusters represented isolates collected in two or more districts. These observations demonstrate that the majority of the genetic clusters are spread throughout the country. The most predominant genetic cluster, representing 21% of the isolates, was identical to the Spanish pandemic clone 23F. In addition, the second largest cluster matched the Spanish-French international clone 9V. These data indicate that the genetic clones 23F and 9V, which are widely spread throughout the world, are the most predominant multidrug-resistant pneumococcal clones in Thailand. Therefore, we conclude that these pandemic clones are primarily responsible for the increase in the prevalence of pneumococcal penicillin resistance in Thailand.
Phylogenetic Analysis of Ara
+
and Ara
−
Burkholderia pseudomallei
Isolates and Development of a Multiplex PCR Procedure for Rapid Discrimination between the Two Biotypes
Authors: Dharakul Tararaj , Tassaneetrithep Boonratn , Trakulsomboon Suwanna , Songsivilai Sirirurg
Published: December 2019

A
Burkholderia pseudomallei
-like organism has recently been identified among some soil isolates of
B. pseudomallei
in an area with endemic melioidosis. This organism is almost identical to
B. pseudomallei
in terms of morphological and biochemical profiles, except that it differs in ability to assimilate
l
-arabinose. These Ara
+
isolates are also less virulent than the Ara
−
isolates in animal models. In addition, clinical isolates of
B. pseudomallei
available to date are almost exclusively Ara
−
. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara
−
and one Ara
+
) and nine soil isolates (five Ara
−
and four Ara
+
) of
B. pseudomallei
were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara
+
biotype was similar to but distinctively different from that of the Ara
−
soil isolates, which were identical to the classical clinical isolates of
B. pseudomallei
. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara
+
or Ara
−
biotypes. The differences were, however, not sufficient for classification into a new species within the genus
Burkholderia
. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara
−
and Ara
+
B. pseudomallei
isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting
B. pseudomallei
in clinical specimens.
Occurrence ofLeishmania infantumParasitemia in Asymptomatic Blood Donors Living in an Area of Endemicity in Southern France
Authors: le Fichoux Yves , Quaranta Jean-François , Aufeuvre Jean-Pierre , Lelievre Alain , Marty Pierre , Suffia Isabelle , Rousseau Deborah , Kubar Joanna
Published: December 2019

Visceral leishmaniosis (VL) due toLeishmania infantum(L. chagasi) is a lethal disease if untreated, but asymptomaticL. infantuminfections have been reported previously. A better understanding of parasite transmission, dissemination, and survival in the human host is needed. The purpose of this study was to assess whetherL. infantumcirculated in peripheral blood of subjects with no history of VL. Sera from 565 blood donors were screened by Western blotting to detectLeishmania-specific antibodies and identify individuals with probable past exposure toLeishmania. Seropositivity was found in 76 donors whose buffy coats were examined by PCR and direct culture. The parasite minicircle kinetoplast DNA was amplified from blood samples of nine donors. Promastigotes were detected by culture in blood samples from nine donors. Only two donors were PCR and culture positive. These results indicate thatL. infantumcirculates intermittently and at low density in the blood of healthy seropositive individuals, who thus appear to be asymptomatic carriers. Implications for the safety of blood transfusion are discussed.
One-Step Reverse Transcriptase PCR Method for Detection of
Borrelia burgdorferi
mRNA in Mouse Lyme Arthritis Tissue Samples
Authors: Limbach F. X. , Jaulhac B. , Piémont Y. , Kuntz J. L. , Monteil H. , Sibilia J.
Published: December 2019

A one-step reverse transcriptase PCR (RT-PCR) method for detection of
Borrelia burgdorferi
mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.
Increased Frequency of Detection of
Ureaplasma urealyticum
and
Mycoplasma genitalium
in AIDS Patients without Urethral Symptoms
Authors: Martinelli F. , Garrafa E. , Turano A. , Caruso A.
Published: December 2019

The roles of
Mycoplasma genitalium
and
Ureaplasma urealyticum
in nongonococcal urethritis are not yet well established. The aim of this study was to determine the presence of these microorganisms in the urethral tracts of 187 human immunodeficiency virus type 1 (HIV-1)-infected male patients with no clinical signs of urethritis. The results indicate that the prevalence of
M. genitalium
and
U. urealyticum
was higher in AIDS patients than in asymptomatic, HIV-1-infected patients and in healthy individuals. The high rate of mycoplasmas and ureaplasmas detected in AIDS patients, in the absence of urethritis, argues against major roles in causing disease at the urethral mucosal level for these microorganisms.
Bacteremia Caused by a Novel Isolate Resembling
Leptotrichia
Species in a Neutropenic Patient
Authors: Patel Jean Baldus , Clarridge Jill , Schuster Mindy S. , Waddington Michael , Osborne Janet , Nachamkin Irving
Published: December 2019

We report a case of
Leptotrichia
species bacteremia in a patient undergoing treatment for acute myelogenous leukemia. Like previously reported
Leptotrichia
species, this is a gram-variable, pleomorphic rod that is catalase negative and utilizes glucose and sucrose. However, it is more fastidious than previously reported isolates of
Leptotrichia
and may represent a novel species.
Improved Methods for Immunoassay of Mycothiol
Authors: Unson Mia D. , Newton Gerald L. , Arnold Karen F. , Davis Charles E. , Fahey Robert C.
Published: December 2019

Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(
N
-maleimidopropionyl)biocytin. The MSH-biotin adduct was then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little as 0.3 fmol of MSH. Methods for biotinylating MSH directly from
Mycobacterium
spp. are described. The MSH-capture ELISA was tested for the detection of
M. avium
seeded in human urine or cerebrospinal fluid samples and for screening mutant
M. smegmatis
strains to detect MSH production.
Differentiation of
Burkholderia
Species by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene and Application to Cystic Fibrosis Isolates
Authors: Segonds Christine , Heulin Thierry , Marty Nicole , Chabanon Gerard
Published: December 2019

Burkholderia cepacia
, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (
B. multivorans
), III, and IV and
B. vietnamiensis. B. gladioli
is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate
Burkholderia
species. This method was applied to 16 type and reference strains of the genus
Burkholderia
and to 51 presumed
B. cepacia
clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12
Burkholderia
type strains tested were differentiated, including
B. cepacia
,
B. multivorans
,
B. vietnamiensis
, and
B. gladioli
, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and
B. pyrrocinia
T
were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes
B. multivorans
T
) and RFLP group 1 (which includes
B. cepacia
genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other
Burkholderia
species (
B. cepacia
genomovar IV or
B. pyrrocinia
,
B. vietnamiensis
, and
B. gladioli
) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each
Burkholderia
species or genomovar in patients with CF.
Molecular Subtyping of
Clostridium perfringens
by Pulsed-Field Gel Electrophoresis To Facilitate Food-Borne-Disease Outbreak Investigations
Authors: Maslanka Susan E. , Kerr Jared G. , Williams Glen , Barbaree James M. , Carson Loretta A. , Miller J. Michael , Swaminathan Bala
Published: December 2019

Clostridium perfringens
is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported. Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals. Serotyping has been used in the past to assist epidemiologic investigations of
C. perfringens
outbreaks. However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents. We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of
C. perfringens
isolates to aid in epidemiologic investigations of food-borne outbreaks. Six restriction endonucleases (
Sma
I,
Apa
I,
Fsp
I,
Mlu
I,
Ksp
I, and
Xba
I) were evaluated with a select panel of
C. perfringens
strains.
Sma
I was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to ∼1,100 kb which provided good discrimination between isolates. Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains. In general, multiple isolates from a single individual had indistinguishable PFGE patterns. Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns. PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by
C. perfringens.
Comparison of the PACE 2 Assay, Two Amplification Assays, and Clearview EIA for Detection of
Chlamydia trachomatis
in Female Endocervical and Urine Specimens
Authors: Lauderdale Tsai-Ling , Landers Lenore , Thorneycroft Ian , Chapin Kimberle
Published: December 2019

Screening for sexually transmitted diseases (STDs) in a greater proportion of sexually active patients has become an accepted protocol by most health care providers. The purpose of this study was to compare the current test methods for detection of
Chlamydia trachomatis
used at the University of South Alabama, the PACE 2 assay (Gen-Probe) and the Clearview EIA (Wampole Laboratories), with two amplification technologies, the AMP CT (Gen-Probe) and LCx (Abbott) assays. In addition, a number of demographic parameters were ascertained by asking questions at the time of examination as well as for health care provider concerns and preferences. One urine and four endocervical swab specimens were collected in random order from 787 female patients attending one of four obstetrics-gynecology clinics. Eighty-seven percent of patients had no STD-related symptoms. Patients were considered positive for
C. trachomatis
if three or more assays (swab and/or urine) were positive. Abbott and Gen-Probe confirmed discrepant results by alternate amplified assays. A total of 66 true-positive specimens were detected by use of the combination of endocervical swabs and urine specimens. After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Sensitivities for the LCx and AMP CT assays with urine specimens were 98 and 81%, respectively. The prevalence of
C. trachomatis
was 8.4%, as determined by amplification technology. Overall, the amplification technologies were the most sensitive methods with either swab (AMP CT assay) or urine (LCx assay) specimens. The PACE 2 assay offered the advantage of a simpler and less expensive assay with acceptable sensitivity. The clearview CT EIA, while yielding a rapid in-office result, had unacceptably low sensitivity. The wide variation in performance with amplification assays with urine specimens as reported in both this study and the literature obviates the need to clarify optimal parameters for this specimen type.
Usefulness of Fatty Acid Composition for Differentiation of
Legionella
Species
Authors: Diogo Alexandra , Veríssimo António , Nobre M. Fernanda , da Costa Milton S.
Published: December 2019

Numerical analysis of fatty acid methyl ester (FAME) profiles of 199 isolates and 76 reference strains, belonging to all validly described species of the genus
Legionella
that can be cultured in laboratory media, was used to differentiate between the species of this genus. With the exception of the strains that autofluoresced red, it was possible to differentiate all the other
Legionella
species. The strains of the species
L. bozemanii
,
L. dumoffii
,
L. feeleii
,
L. gormanii
,
L. maceachernii
,
L. micdadei
, and
L. quinlivanii
did not form single clusters, showing some degree of variability in the fatty acid compositions. The strains of the blue-white autofluorescent species had very similar fatty acid compositions and were difficult to distinguish from each other. Nine isolates had fatty acid profiles unlike those of any of the validly described species and may represent different FAME groups of known species or undescribed
Legionella
species. The method used in this study was useful for screening and discriminating large number of isolates of
Legionella
species. Moreover, the results obtained can be included in a database of fatty acid profiles, leading to a more accurate automatic identification of
Legionella
isolates.
Rapid Detection of Respiratory Viruses by Shell Vial Assay Using Simultaneous Culture of HEp-2, LLC-MK2, and MDCK Cells in a Single Vial
Authors: Navarro-Marí José M. , Sanbonmatsu-Gámez Sara , Pérez-Ruiz Mercedes , De La Rosa-Fraile Manuel
Published: December 2019

A shell vial assay with simultaneous culture of HEp-2, LLC-MK2, and MDCK cell lines in a single tube (CoHLM SV assay) was compared with traditional tube culture (TC) for the detection of the main respiratory viruses in 358 nasal wash specimens. A total of 170 strains were isolated from 168 virus-positive samples. A total of 94.1% of the strains (160 strains; 128 respiratory syncytial viruses and 32 other viruses) were detected by the CoHLM SV assay in 48 h, whereas 98.2% of the strains (167 strains; 132 respiratory syncytial viruses and 35 other viruses) were detected by TC in a mean time of 6 days. The CoHLM SV assay may be useful for the rapid detection of respiratory viruses.
Inactivation of
Mycobacterium tuberculosis
for DNA Typing Analysis
Authors: Bemer-Melchior P. , Drugeon H. B.
Published: December 2019

DNA fingerprinting analysis of
Mycobacterium tuberculosis
is used for epidemiological studies and the control of laboratory cross-contamination. Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens. The technique ensures the inactivation of
M. tuberculosis
before DNA extraction without the loss of DNA integrity. The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months.
Prototypal Diarrheagenic Strains of
Hafnia alvei
Are Actually Members of the Genus
Escherichia
Authors: Janda J. Michael , Abbott Sharon L. , Albert M. John
Published: December 2019

We analyzed five bacterial strains, designated 19982, 9194, 10457, 10790, and 12502, that were isolated from stool specimens of individuals with diarrheal illness by the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (M. J. Albert, S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K. Alam, I. Kabir, and R. Robins-Browne, J. Med. Microbiol. 37:310–314, 1992). The strains were initially identified as
Hafnia alvei
with a commercial identification system and were reported to contain the
eae
gene of enteropathogenic
Escherichia coli
. Results of conventional biochemical analyses, testing of susceptibility to cephalothin, lysis by a
Hafnia
-specific phage, and amplification of the outer membrane protein gene
phoE
with species-specific primers support the identification of these strains as members of the genus
Escherichia
rather than
Hafnia alvei
. These strains varied from typical
E. coli
strains by their inability to produce acid from lactose or
d
-sorbitol and failure to elaborate the enzyme β-
d
-glucuronidase. PCR analysis confirmed previous findings that the strains were positive for the
eae
gene and negative for other virulence markers present among recognized categories of diarrheagenic
E. coli
. Our findings support the hypothesis that these strains are a new category of diarrheagenic isolates belonging to the genus
Escherichia
and illustrate the importance of using multiple methodologies when identifying new bacterial agents of diarrheal disease.
Involvement of Enterotoxins G and I in Staphylococcal Toxic Shock Syndrome and Staphylococcal Scarlet Fever
Authors: Jarraud Sophie , Cozon Grégoire , Vandenesch François , Bes Michèle , Etienne Jerome , Lina Gerard
Published: December 2019

We investigated the involvement of the recently described staphylococcal enterotoxins G and I in toxic shock syndrome. We reexamined
Staphylococcus aureus
strains isolated from patients with menstrual and nonmenstrual toxic shock syndrome (nine cases) or staphylococcal scarlet fever (three cases). These strains were selected because they produced none of the toxins known to be involved in these syndromes (toxic shock syndrome toxin 1 and enterotoxins A, B, C, and D), enterotoxin E or H, or exfoliative toxin A or B, despite the fact that superantigenic toxins were detected in a CD69-specific flow cytometry assay measuring T-cell activation. Sets of primers specific to the enterotoxin G and I genes (
seg
and
sei
, respectively) were designed and used for PCR amplification. All of the strains were positive for
seg
and
sei
. Sequence analysis confirmed that the PCR products, corresponded to the target genes. We suggest that staphylococcal enterotoxins G and I may be capable of causing human staphylococcal toxic shock syndrome and staphylococcal scarlet fever.
Comparison of the Second-Generation Digene Hybrid Capture Assay with the Branched-DNA Assay for Measurement of Hepatitis B Virus DNA in Serum
Authors: Ho Stephen K. N. , Chan Tak Mao , Cheng Ignatius K. P. , Lai Kar Neng
Published: December 2019

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 ± 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 × [HBV DNA by bDNA (megaequivalents per milliliter)]
0.866
. The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants.
Value of Enterobacterial Repetitive Intergenic Consensus PCR for Study of
Pasteurella multocida
Strains Isolated from Mouths of Dogs
Authors: Loubinoux Julien , Lozniewski Alain , Lion Christine , Garin Daniel , Weber Michèle , Le Faou Alain E.
Published: December 2019

Fifty-six
Pasteurella multocida
strains (40
P. multocida
subsp.
septica
and 16
P. multocida
subsp.
multocida
strains) isolated from the mouths of 56 dogs among the 134 living in a French canine military training center (132e Groupe Cynophile de l’Armée de Terre, Suippes, France) were studied by use of enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and restriction fragment length polymorphism (RFLP) techniques. Both techniques showed genomic heterogeneity of the strains studied. However, RFLP was more discriminatory than ERIC-PCR for differentiating
P. multocida
strains. All but three pairs of strains were discriminated by RFLP, suggesting a limited circulation of strains between these dogs living in proximity. Although ERIC-PCR is easier and faster to perform, it cannot be recommended for epidemiological studies of
P. multocida
strains.
Phialemonium
Fungemia: Two Documented Nosocomial Cases
Authors: Guarro Josep , Nucci Marcio , Akiti Tiyomi , Gené Josepa , Cano Josep , Barreiro M. Da Gloria C. , Aguilar Carme
Published: December 2019

Two fungal isolates recovered from the blood of two immunosuppressed patients are described as
Phialemonium curvatum
. One patient died, while the other, who was infected with
Exophiala jeanselmei
at the same time, survived after successful treatment with itraconazole. Analysis of internal transcribed spacer sequences demonstrated that the isolates belonged to the same strain and that the source of infection was probably a catheter. The taxonomic position of
P. curvatum
is discussed, and
Phialemonium dimorphosporum
is considered a synonym. The in vitro inhibitory activities of six antifungal agents (amphotericin B, itraconazole, ketaconazole, miconazole, flucytosine, and fluconazole) were determined against seven isolates of
Phialemonium
. Except for flucytosine, all of them were remarkably effective.
Phialemonium
should be added to the list of potential causes of nosocomial fungemia in cancer patients.
Molecular Epidemiological Study ofHaemophilus influenzaeSerotype b Strains Obtained from Children with Meningitis in Japan
Authors: Mitsuda Toshihiro , Kuroki Haruo , Ishikawa Nobuyasu , Imagawa Tomoyuki , Ito Schuichi , Miyamae Takako , Mori Masaaki , Uehara Suzuko , Yokota Shumpei
Published: December 2019

We report an epidemiological study of 30Haemophilus influenzaeserotype b (Hib) strains derived from the cerebrospinal fluid of children with meningitis. The Hib strains were biotyped, tested for β-lactamase production, and genotyped by long PCR-ribotyping, random amplified polymorphic DNA (RAPD) analysis, and genomic DNA restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). The phenotypic study characterized 22 of the strains (73%) as biotype I. A genotypic study using long PCR-ribotyping withHaeIII restriction digestion showed no polymorphisms among these 30 Hib strains, but RAPD analysis with two sets of primers demonstrated two distinctive subtypes: one typical of the strains of biotype group II and the second characteristic of the strains of biotype groups I and IV. Each RAPD group was subtyped into several genotypic groups by PFGE-RFLP withSmaI digestion. The genotyping of clinically isolated Hib strains may help to elucidate transmission routes in community infections, endemicity, and the reasons for vaccine failure.