bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Epidemiology of Human Group A Rotavirus Infections in the United Kingdom between 1995 and 1998
Authors: Iturriza-Gómara Miren , Green Jon , Brown David W. G. , Ramsay Mary , Desselberger Ulrich , Gray James J.
Published: December 2019

The G and P types of 2,912 rotavirus-positive fecal specimens collected from eight geographical areas of the United Kingdom between 1995 and 1998 were determined by reverse transcription-PCR. Although 15 different G-P combinations were identified, G1P[8], G2P[4], G3P[8], and G4P[8] strains constituted 95% of all the rotaviruses typed. Other genotypes included G9P[6] and G9P[8], which were first identified in the United Kingdom in 1995, or other uncommon G and/or P types of strains that may have had an animal origin. Unusual combinations of G1 or G4 with P[4] and G2 with P[8] which may have arisen by reassortment between human strains were also identified. G1P[8] was the genotype most frequently found (57 to 87%) in each season, followed by G2P[4] in the 1995–1996 (18%) and 1997–1998 (16%) seasons, although the incidence of infection with this virus decreased significantly to 2% during the 1996–1997 season. Significant differences were seen in the distributions of G1P[8], G2P[4], and G9P[8] strains between children and adults, in the temporal distributions of G4P[8] and G9P[8] strains within a season, and in the geographical distributions of each of the four most common genotypes from one season to the next.
Molecular Characterization and Diagnostic Value of
Taenia solium
Low-Molecular-Weight Antigen Genes
Authors: Sako Yasuhito , Nakao Minoru , Ikejima Takashi , Piao Xian Zhi , Nakaya Kazuhiro , Ito Akira
Published: December 2019

Neurocysticercosis (NCC) caused by infection with the larvae of
Taenia solium
is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using an
Escherichia coli
expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.
Distinct Differences in Repertoires of Low-Molecular-Mass Secreted Antigens of
Mycobacterium avium
Complex and
Mycobacterium tuberculosis
Authors: Olsen Ingrid , Reitan Liv J. , Wiker Harald G.
Published: December 2019

Antigens in a 4-week-old culture filtrate (CF) of
Mycobacterium avium
subsp.
avium
were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of
Mycobacterium tuberculosis
with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the
M. avium
subsp.
avium
CF. In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of
M. avium
subsp.
avium
which was highly homologous to Rv0455c of
M. tuberculosis
. The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of
M. avium
subsp.
avium
,
Mycobacterium avium
subsp.
paratuberculosis
,
Mycobacterium intracellulare
, and
Mycobacterium scrofulaceum
but was not detected in
M. tuberculosis
. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species.
Variable Numbers of TTC Repeats inMycobacterium lepraeDNA from Leprosy Patients and Use in Strain Differentiation
Authors: Shin Yoo-Chul , Lee Hyejon , Lee Hyeyoung , Walsh Gerald P. , Kim Joo-Deuk , Cho Sang-Nae
Published: December 2019

Strain differentiation ofMycobacterium lepraewould be of great value for epidemiological investigation to identify the infectious sources of leprosy, to understand transmission patterns, and to distinguish between relapse and reinfection. From theM. lepraegenome sequence database, TTC DNA repeats were identified. Primer sets designed to amplify the region flanking TTC repeats revealed PCR products of different sizes, indicating that the number of repeats at each locus may be variable amongM. lepraestrains. The TTC repeats were not found inMycobacterium tuberculosis,Mycobacterium avium,Mycobacterium marinum, or human tissues, which indicated their specificity toM. leprae. Sequence analysis of the TTC repeat region in each of theM. lepraestrains showed a variation of 10 to 37 repeats. In theM. lepraestrains of 34 multibacillary patients at Cebu, Philippines,M. lepraewith 24 and 25 TTC repeats was most common, and this was followed by strains with 14, 15, 20, 21, and 28 repeats. This study thus indicates that there are variable numbers of TTC repeats in a noncoding region ofM. lepraestrains and that the TTC region may be useful for strain differentiation for epidemiological investigations of leprosy.
Genetic Relatedness within Serotypes of Penicillin-Susceptible
Streptococcus pneumoniae
Isolates
Authors: Overweg Karin , Bogaert Debby , Sluijter Marcel , Yother Janet , Dankert Jacob , de Groot Ronald , Hermans Peter W. M.
Published: December 2019

The molecular epidemiological characteristics of all
Streptococcus pneumoniae
strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (
n
= 27), Thailand (
n
= 9), The Netherlands (
n
= 8), and Denmark (
n
= 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.
Comparative Genotyping of
Candida albicans
Bloodstream and Nonbloodstream Isolates at a Polymorphic Microsatellite Locus
Authors: Dalle Frédéric , Franco Norelie , Lopez José , Vagner Odile , Caillot Denis , Chavanet Pascal , Cuisenier Bernadette , Aho Serge , Lizard Sarab , Bonnin Alain
Published: December 2019

Molecular typing studies have shown that the predominant form of reproduction of
Candida albicans
is clonal and that, in a majority of situations, persistent or recurrent infections are due to a unique strain. Characterization of distinct subpopulations and correlation with clinical features may thus be important to understanding the pathogenesis of candidiasis. In a clonal model, a unique polymorphic marker may identify populations with different biological properties. We therefore compared 48 bloodstream isolates and 48 nonbloodstream matched strains of
C. albicans
at the elongation factor 3-encoding gene (CEF3) polymorphic microsatellite locus of
C. albicans
. Sizing of the alleles was performed by automated capillary electrophoresis. A new, 137-bp allele was characterized, and seven nondescribed combinations were observed, resulting in 15 and 11 distinct CEF3 profiles in bloodstream and control strains, respectively. Genotypes 126-135, 130-136, and 131-131 accounted for 60.4% of both bloodstream and control strains. Four bloodstream isolates but no control strains displayed the 135-135 combination. None of the other genotypes was present at an increased frequency in bloodstream isolates. Bloodstream and nonbloodstream strains of
C. albicans
thus have a heterogeneous structure at the CEF3 locus, with three major and multiple minor allelic combinations.
Infection of Hickman Catheter by
Pseudomonas
(formerly
Flavimonas
)
oryzihabitans
Traced to a Synthetic Bath Sponge
Authors: Marı́n Mercedes , Garcı́a de Viedma Darı́o , Martı́n-Rabadán Pablo , Rodrı́guez-Créixems Marta , Bouza Emilio
Published: December 2019

Pseudomonas
(formerly
Flavimonas
)
oryzihabitans
is an uncommon pathogen that may cause catheter-associated infections. Although it has occasionally been isolated from the environment, the source of human infection has not previously been documented. We describe an AIDS patient who developed
Pseudomonas oryzihabitans
bacteremia due to colonization of a Hickman catheter. The patient reported having strictly followed the recommendations for catheter hygiene. The only flaw detected was the use of a synthetic bath sponge in the shower. The sponge was cultured and yielded
P. oryzihabitans
among other nonfermentative, gram-negative bacilli. To determine the prevalence of
P. oryzihabitans
in sponges, we cultured 15 samples from unrelated households. The microorganism was isolated from 3 of the 15 samples. Molecular typing by arbitrarily primed PCR (AP-PCR) was performed with the environmental and clinical isolates. Three different profiles were obtained for the six isolates analyzed from the patient's sponge. The strain from the AIDS patient was identical to one of those from his sponge and was different from all the remaining strains. The AP-PCR typing results were subsequently confirmed by pulsed-field gel electrophoresis. It can be concluded that sponges are occasionally colonized by
P. oryzihabitans
. For the first time a probable source of an indwelling catheter contamination with this bacterium has been found. Patients carrying these devices should avoid using sponge-like materials, as these are suitable environments for nonfermentative, gram-negative bacilli.
Cost-Effective and Rapid Presumptive Identification of Gram-Negative Bacilli in Routine Urine, Pus, and Stool Cultures: Evaluation of the Use of CHROMagar Orientation Medium in Conjunction with Simple Biochemical Tests
Authors: Ohkusu Kiyofumi
Published: December 2019

The algorithm for a new identification system was designed on the basis of colony color and morphology on CHROMagar Orientation medium in conjunction with simple biochemical tests such as indole (IND), lysine decarboxylase (LDC), and ornithine decarboxylase (ODC) utilization tests with gram-negative bacilli isolated from urine samples as well as pus, stool, and other clinical specimens by the following colony characteristics, biochemical reactions, and serological results: pinkish to red, IND positive (IND
+
),
Escherichia coli
; metallic blue, IND
+
, LDC
+
, and ODC negative (ODC
−
),
Klebsiella oxytoca
; IND
+
, LDC
−
, and ODC
+
,
Citrobacter diversus
; IND
+
or IND
−
, LDC
−
, and ODC
−
,
Citrobacter freundii
; IND
−
, LDC
+
, and ODC
+
,
Enterobacter aerogenes
; IND
−
, LDC
−
, and ODC
+
,
Enterobacter cloacae
; IND
−
, LDC
+
, and ODC
−
,
Klebsiella pneumoniae
; diffuse brown and IND
+
,
Morganella morganii
; IND
−
,
Proteus mirabilis
; aqua blue,
Serratia marcescens
; bluish green and IND
+
,
Proteus vulgaris
; transparent yellow-green, serology positive,
Pseudomonas aeruginosa
; clear and serology positive,
Salmonella
sp.; other colors and reactions, the organism was identified by the full identification methods. The accuracy and cost-effectiveness of this new system were prospectively evaluated. During an 8-month period, a total of 345 specimens yielded one or more gram-negative bacilli. A total of 472 gram-negative bacillus isolates were detected on CHROMagar Orientation medium. For 466 of the isolates (98.7%), no discrepancies in the results were obtained on the basis of the identification algorithm. The cost of identification of gram-negative bacilli during this period was reduced by about 70%. The results of this trial for the differentiation of the most commonly encountered gram-negative pathogens in clinical specimens with the new algorithm were favourable in that it permitted reliable detection and presumptive identification. In addition, this rapid identification system not only significantly reduced costs but it also improved the daily work flow within the clinical microbiology laboratory.
PCR-Restriction Enzyme Analysis of a Bone Marrow Isolate from a Human Immunodeficiency Virus-Positive Patient Discloses Polyclonal Infection with Two
Mycobacterium avium
Strains
Authors: Oliveira R. S. , Sircili M. P. , Ueki S. Y. M. , Telles M. A. S. , Schnabel B. , Briones M. R. S. , Leão S. C.
Published: December 2019

Polyclonal infection by
Mycobacterium avium
was detected by
hsp65
PCR-restriction enzyme analysis (PRA) in a bone marrow isolate from an AIDS patient. Two
M. avium
strains, differing in colony morphology, PRA
Hae
III digestion pattern, insertion element (IS)
1245
amplification, and restriction fragment length polymorphism fingerprints with IS
1245
and IS
1311
probes, were isolated.
Preliminary Evaluation of a Semisolid Agar Antifungal Susceptibility Test for Yeasts and Molds
Authors: Provine Harriet , Hadley Susan
Published: December 2019

This report presents a semisolid agar antifungal susceptibility (SAAS) method for the rapid susceptibility screening of yeasts and molds. The reproducibility and accuracy of the SAAS method were assessed by comparing the MICs of amphotericin B and fluconazole obtained for 10 candidate quality control (QC) American Type Culture Collection yeast strains in ≥15 replicates with those found by six independent laboratories using the National Committee for Clinical Laboratory Standards (NCCLS) M27-P broth macrodilution method (M. A. Pfaller et al., J. Clin. Microbiol. 33:1104–1107, 1995). Overall, 96% of MICs for both drugs fell within 1 log
2
dilution of the modal MIC for each strain. The MICs for amphotericin B showed 99% agreement with the NCCLS proposed QC ranges within 1 log
2
dilution. Likewise, the MICs for fluconazole at ≥75% growth reduction showed 99% agreement for seven strains. Three strains,
Candida albicans
ATCC 24333 and ATCC 76615 and
Candida tropicalis
ATCC 750, showed a less sharp fluconazole endpoint at ≥75% growth reduction, but at >50% growth reduction, the agreement was 98% within 1 log
2
dilution of the proposed range. The MIC agreement within the proposed range for the suggested QC strains
Candida parapsilosis
ATCC 22019 and
Candida krusei
ATCC 6258 was 100% for fluconazole and 100% within 1 log
2
dilution of the proposed range for amphotericin B. The SAAS method demonstrated the susceptibility or resistance of 25 clinical isolates of filamentous fungi such as
Aspergillus fumigatus
to amphotericin B, itraconazole, and fluconazole, usually within 48 h. Although the results are preliminary, this SAAS method is promising as a rapid and cost-effective screen and is worthy of concerted investigation.
Detection of Extended-Spectrum β-Lactamases in Clinical Isolates of
Enterobacter cloacae
and
Enterobacter aerogenes
Authors: Tzelepi Eva , Giakkoupi Panagiota , Sofianou Danai , Loukova Veneta , Kemeroglou Anastassia , Tsakris Athanassios
Published: December 2019

The aim of the present study was to investigate the frequency of extended-spectrum β-lactamases (ESBLs) in a consecutive collection of clinical isolates of
Enterobacter
spp. The abilities of various screening methods to detect ESBLs in enterobacters were simultaneously tested. Among the 68 consecutive isolates (56
Enterobacter cloacae
and 12
Enterobacter aerogenes
isolates) that were analyzed for β-lactamase content, 21 (25 and 58%, respectively) possessed transferable ESBLs with pIs of 8.2 and phenotypic characteristics of SHV-type enzymes, 8 (14.3%) of the
E. cloacae
isolates produced a previously nondescribed, clavulanate-susceptible ESBL that exhibited a pI of 6.9 and that conferred a ceftazidime resistance phenotype on
Escherichia coli
transconjugants, and 2
E. cloacae
isolates produced both of these enzymes. Among the total of 31 isolates that were considered ESBL producers, the Vitek ESBL detection test was positive for 2 (6.5%) strains, and the conventional double-disk synergy test (DDST) with amoxicillin-clavulanate and with expanded-spectrum cephalosporins and aztreonam was positive for 5 (16%) strains. Modifications of the DDST consisting of closer application of the disks (at 20 instead of 30 mm), the use of cefepime, and the use of both modifications increased the sensitivity of this test to 71, 61, and 90%, respectively. Of the 37 isolates for which isoelectric focusing failed to determine ESBLs, the Vitek test was false positive for 1 isolate and the various forms of DDSTs were false-positive for 3 isolates.
Evaluation of the COBAS AMPLICOR CMV MONITOR Test for Detection of Viral DNA in Specimens Taken from Patients after Liver Transplantation
Authors: Sia Irene G. , Wilson Jennie A. , Espy Mark J. , Paya Carlos V. , Smith Thomas F.
Published: December 2019

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.
Serologic Testing for
Trypanosoma cruzi
: Comparison of Radioimmunoprecipitation Assay with Commercially Available Indirect Immunofluorescence Assay, Indirect Hemagglutination Assay, and Enzyme-Linked Immunosorbent Assay Kits
Authors: Leiby David A. , Wendel Silvano , Takaoka Deise T. , Fachini Roberta M. , Oliveira Lea C. , Tibbals Melinda A.
Published: December 2019

The radioimmunoprecipitation assay (RIPA) has been used as a confirmatory test in several ongoing and published studies of
Trypanosoma cruzi
in blood donors in the United States. Despite its use as a confirmatory test, few studies are available comparing RIPA to commercially available serologic test methods. Thus, we compared RIPA with two indirect hemagglutination assays (Biolab Diagnostica SA, São Paulo, Brazil; Hemagen Diagnostics, Inc., Waltham, Mass.) and four different enzyme-linked immunosorbent assays (Abbott Laboratories, Abbott Park, Ill.; Embrabio, São Paulo, Brazil; Organon Teknika, São Paulo, Brazil; and Gull Laboratories, Salt Lake City, Utah) using a panel of 220 serum specimens from Brazilian blood donors with a range of
T. cruzi
antibody titers as determined by indirect immunofluorescence assay (IFA). A titer of 1:20 was used as the baseline for seropositivity. All IFA-negative serum specimens (
n
= 19) were nonreactive on all tests. At a titer of 1:20 (
n
= 9), reactivity rates varied considerably among the tests, with only the RIPA and the Organon and Gull assays identifying reactive specimens. For specimens at a 1:40 titer (
n
= 35), most assays identified at least 32 of 35 (91%) specimens as reactive, but the Biolab assay only identified 24 (69%). At higher titers (1:80,
n
= 56; 1:160,
n
= 101) the assays were comparable, with the exception of the Biolab assay, demonstrating rates of agreement with IFA of ≥98%. Overall, when compared with several other test formats, RIPA demonstrated equivalent or superior rates of agreement with IFA-positive specimens across all titers examined. In particular, at titers of >1:40, the RIPA compared favorably with other test methods currently in use, supporting its application as a confirmatory test, particularly in a research setting.
Development of a Real-Time Quantitative Assay for Detection of Epstein-Barr Virus
Authors: Niesters Hubert G. M. , van Esser Joost , Fries Edwin , Wolthers Katja C. , Cornelissen Jan , Osterhaus Albert D. M. E.
Published: December 2019

With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10
7
copies of DNA per ml using two sample preparation methods based on absorption. A precision study yielded an average coefficient of variation for both methods of less than 12%, with a coefficient of regression for the standard curve of a minimum of 0.98. We detected EBV DNA in 19.2% of plasma samples from immunosuppressed solid-organ transplant patients without symptoms of EBV infections with a mean load of 440 copies per ml. EBV DNA could be detected in all transplant patients diagnosed with posttransplant lymphoproliferative disorder, with a mean load of 544,570 copies per ml. No EBV DNA could be detected in healthy individuals in nonimmunosuppressed control groups and a mean of 6,400 copies per ml could be detected in patients with infectious mononucleosis. Further studies revealed that the inhibitory effect of heparinized plasma could be efficiently removed by use of an extraction method with Celite as the absorbent.
Survival of Enterococci and Staphylococci on Hospital Fabrics and Plastic
Authors: Neely Alice N. , Maley Matthew P.
Published: December 2019

The transfer of gram-positive bacteria, particularly multiresistant
Staphylococcus aureus
(MRSA) and vancomycin-resistant enterococci (VRE), among patients is a growing concern. One critical aspect of bacterial transfer is the ability of the microorganism to survive on various common hospital surfaces. The purpose of this study was to determine the survival of 22 gram-positive bacteria (vancomycin-sensitive and -resistant enterococci and methicillin-sensitive and -resistant staphylococci) on five common hospital materials: smooth 100% cotton (clothing), 100% cotton terry (towels), 60% cotton–40% polyester blend (scrub suits and lab coats), 100% polyester (privacy drapes), and 100% polypropylene plastic (splash aprons). Swatches were inoculated with 10
4
to 10
5
CFU of a microorganism, assayed daily by placing the swatches in nutritive media, and examining for growth after 48 h. All isolates survived for at least 1 day, and some survived for more than 90 days on the various materials. Smaller inocula (10
2
) survived for shorter times but still generally for days. Antibiotic sensitivity had no consistent effect on survival. The long survival of these bacteria, including MRSA and VRE, on commonly used hospital fabrics, such as scrub suits, lab coats, and hospital privacy drapes, underscores the need for meticulous contact control procedures and careful disinfection to limit the spread of these bacteria.
Correlation of Oxacillin MIC with
mecA
Gene Carriage in Coagulase-Negative Staphylococci
Authors: Hussain Zafar , Stoakes Luba , Massey Viki , Diagre Deb , Fitzgerald Viivi , El Sayed Sameer , Lannigan Robert
Published: December 2019

The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from ≥4 mg/liter to ≥0.5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the
mecA
gene, 493 CoNS of 11 species were tested. The presence of the
mecA
gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0.125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with
mecA
as methicillin resistant and strains of
Staphylococcus epidermidis
,
S. haemolyticus
, and
S. hominis
without
mecA
as methicillin susceptible. The breakpoint of ≥0.5 mg/liter was not specific for
S. cohnii
,
S. lugdunensis
,
S. saprophyticus
,
S. warneri
, and
S. xylosus
, in that it categorized 70 of 74 strains of these species without
mecA
(94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with
mecA
but is not specific for strains of certain species of CoNS without
mecA
.
Molecular Epidemiology of Rabies Virus Isolates from Israel and Other Middle- and Near-Eastern Countries
Authors: David D. , Yakobson B. , Smith J. S. , Stram Y.
Published: December 2019

A total of 226 isolates of rabies virus from different areas of Israel, including three human isolates and one sample from South Lebanon were identified between 1993 and 1998 by direct immunofluorescence using monoclonal antibodies to the viral nucleoprotein (N). An epidemiological survey based on nucleotide sequence analysis of 328 bp from the C terminus of the N coding region and the noncoding region between the nucleoprotein and the phosphoprotein (NS gene) was performed. Phylogenetic analysis of the isolates from Israel showed that they were related geographically, but not according to host species. Five variants, related groups distributed among four geographical regions, were identified. In each region, rabies virus was isolated from more than one animal species. A comparison of the sequence analysis of rabies virus samples from the rest of world revealed a 2-nucleotide change that distinguished the Middle East variants from the rest.
Unusual Case of
Acanthamoeba polyphaga
and
Pseudomonas aeruginosa
Keratitis in a Contact Lens Wearer from Gauteng, South Africa
Authors: Dini L. A. , Cockinos C. , Frean J. A. , Niszl I. A. , Markus M. B.
Published: December 2019

Acanthamoeba
species can cause a chronic, progressive ulcerative keratitis of the eye which is not responsive to the usual antimicrobial therapy and is frequently mistaken for stromal herpes keratitis. An unusual case of coinfection with
Acanthamoeba polyphaga
and
Pseudomonas aeruginosa
as causes of corneal keratitis in a contact lens wearer from Gauteng, South Africa, is reported. These two pathogens have previously been assumed to be selectively exclusive. Cysts of the isolated acanthameba tolerated an incubation temperature of 40°C, indicating a pathogenic species. This case highlights the importance of culture methods in the diagnosis of corneal infection and the choice of treatment regimen. The patient's history of careless contact lens-disinfecting habits emphasizes the need to adhere strictly to recommended methods of contact lens care.
Thyrotoxicosis Induced by Thyroid Involvement of Disseminated
Aspergillus fumigatus
Infection
Authors: Hornef Mathias W. , Schopohl Jochen , Zietz Christian , Hallfeldt Klaus K. J. , Roggenkamp Andreas , Gärtner Roland , Heesemann Jürgen
Published: December 2019

Aspergillus fumigatus
is increasingly recognized as an important nosocomial pathogen in severely immunocompromised patients. Infection is difficult to diagnose antemortem and typically has a fatal outcome. Here we report the case of a cardiac transplant recipient with disseminated
A. fumigatus
infection which clinically presented as thyrotoxicosis due to massive involvement of the thyroid gland.
Growth Competition between
Candida dubliniensis
and
Candida albicans
under Broth and Biofilm Growing Conditions
Authors: Kirkpatrick W. R. , Lopez-Ribot J. L. , Mcatee R. K. , Patterson T. F.
Published: December 2019

Seven isolates each of
Candida albicans
and
Candida dubliniensis
were paired (11 pairs) and examined for competitive interaction. Equal numbers of CFU of each competitor were inoculated into Sabouraud dextrose broth and incubated at 37°C with vigorous shaking under conditions favorable to either broth or biofilm growth. Surviving proportions of each competitor were calculated from the broth culture at 24 and 96 h and the biofilm culture at 96 h, with species differentiation done on CHROMagar Candida medium.
C. albicans
had a competitive advantage over
C. dubliniensis
in broth culture and under biofilm growing conditions; however, with the presence of a supporting structure for biofilm formation,
C. dubliniensis
was able to better withstand the competitive pressures from
C. albicans
.
Temperature-Sensitive Strain ofCryptococcus neoformansProducing Hyphal Elements in a Feline Nasal Granuloma
Authors: Bemis D. A. , Krahwinkel D. J. , Bowman L. A. , Mondon P. , Kwon-Chung K. J.
Published: December 2019

We report the isolation of a temperature-sensitive, serotype A, mating type α strain ofCryptococcus neoformansfrom a case of nasal cryptococcosis in a cat. The strain grew extremely slowly at 35°C and failed to grow at 37°C in vitro. Histopathological sections of the infected tissue revealed yeast cells producing hyphae up to several hundred micrometers in length, in addition to numerous encapsulated yeast cells typical ofC. neoformans. The cultures grown on yeast extract-peptone-glucose agar at 35°C also produced some yeast cells with germ tube-like hyphal elements up to 100 μm in length.
Prevalence and Characterization of Shiga Toxin-Producing
Escherichia coli
Isolated from Cattle, Food, and Children during a One-Year Prospective Study in France
Authors: Pradel Nathalie , Livrelli Valérie , De Champs Christophe , Palcoux Jean-Bernard , Reynaud Alain , Scheutz Flemming , Sirot Jacques , Joly Bernard , Forestier Christiane
Published: December 2019

During a 1-year survey of Shiga toxin-producing
Escherichia coli
(STEC) prevalence in central France, 2,143 samples were investigated by PCR for Shiga toxin-encoding genes. A total of 330 (70%) of 471 fecal samples collected from healthy cattle at the Clermont-Ferrand slaughterhouse, 47 (11%) of 411 beef samples, 60 (10%) of 603 cheese samples, and 19 (3%) of 658 stool specimens from hospitalized children with and without diarrhea were positive for the
stx
gene(s). A STEC strain was isolated from 34% (162 of 471) of bovine feces, 4% (16 of 411) of beef samples, 1% (5 of 603) of cheese samples, and 1.5% (10 of 658) of stool specimens. Of the 220 STEC strains isolated, 34 (15%) harbored the
stx
1
gene, 116 (53%) harbored the
stx
2
gene, and 70 (32%) carried both the
stx
1
and
stx
2
genes. However, 32 (14.5%) were not cytotoxic for Vero cells. The
eae
gene, found in 12 (5%) of the 220 strains, was significantly associated with the
stx
1
gene and with isolates from children. Sequences homologous to
ehxA
were found in 102 (46%) of the 220 strains. Thirteen serotypes, OX3:H2, O113:H21, O113:H4, OX3:H21, O6:H10, OX178:H19, O171:H2, O46:H38, O172:H21, O22:H16, O91:H10, O91:H21, and O22:H8, accounted for 102 (55%) of 186 typeable isolates, and only one strain (0.5% of the 186 STEC isolates from cattle), belonged to the O157:H7 serotype. We showed that the majority of the STEC isolates from cattle, beef, and cheese were not likely to be pathogenic for humans and that the STEC strains isolated from children in this study were probably not responsible for diarrheal disease. Finally, the strains associated with hemolytic-uremic syndrome in the same geographical area were shown to belong to particular subsets of the STEC population found in the bovine reservoir.
Epidemiology of Astrovirus Infection in Young Children Hospitalized with Acute Gastroenteritis in Melbourne, Australia, over a Period of Four Consecutive Years, 1995 to 1998
Authors: Mustafa Huseyin , Palombo Enzo A. , Bishop Ruth F.
Published: December 2019

The incidence of astrovirus infection in children less than 5 years of age hospitalized with acute gastroenteritis in Melbourne, Australia, from 1995 to 1998 was determined. Astrovirus was detected in 40 of 449 specimens tested by Northern hybridization, and astrovirus infection was confirmed by reverse transcription-PCR with or without culture in CaCO-2 cells. This represented 3.0% (40 of 1,327) of all children tested for enteric pathogens, including viral, bacterial, and parasitic pathogens, over the survey period. The incidences of astrovirus infection in each year were 4.4% (1995), 2.2% (1996), 3.9% (1997), and 1.4% (1998). In 1995 and 1997, the incidences of astrovirus infection were greater than the incidence of infection with all individual bacterial pathogens and were either greater than or equal to the incidence of adenovirus infection. Astrovirus exhibited an unusual biennial winter peak of incidence that correlated with a greater incidence of serotype 1 virus and an increased rate of hospitalization of children aged 6 to 12 months. Uncommon (serotype 2 and 4) and rare (serotype 8) serotypes were detected during the survey period. Genetic analysis of ORF2 (which encodes the astrovirus capsid precursor) of Melbourne isolates showed nucleotide sequence variation from year to year. This was not accompanied by significant amino acid substitutions. However, geographical variation was apparent by comparison of Melbourne astrovirus isolates with prototype strains identified in the United Kingdom.
Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases
Authors: Roth Andreas , Reischl Udo , Streubel Anna , Naumann Ludmila , Kroppenstedt Reiner M. , Habicht Marion , Fischer Marga , Mauch Harald
Published: December 2019

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only
Gordonia terrae
was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with
Hae
III and
Cfo
I restriction analysis. It yielded 58
Hae
III patterns, of which 49 (84%) were unique on the species level. Hence,
Hae
III digestion together with
Cfo
I results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in
Mycobacterium intracellulare
and
Mycobacterium kansasii
, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the
Mycobacterium avium
complex or
Mycobacterium chelonae-Mycobacterium abscessus
) that were successfully separated using additional enzymes (
Taq
I,
Msp
I,
Dde
I, or
Ava
II). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including
M. abscessus
,
M. chelonae
,
Mycobacterium farcinogenes
,
Mycobacterium fortuitum
,
Mycobacterium peregrinum
, and
Mycobacterium senegalense
(with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.
Efficacy of Antimicrobial Treatments and Vaccination Regimens for Control of Porcine Reproductive and Respiratory Syndrome Virus and
Streptococcus suis
Coinfection of Nursery Pigs
Authors: Halbur P. , Thanawongnuwech R. , Brown G. , Kinyon J. , Roth J. , Thacker E. , Thacker B.
Published: December 2019

Seventy-six, crossbred, porcine reproductive and respiratory syndrome virus (PRRSV)-free pigs were weaned at 12 days of age and randomly assigned to seven groups of 10 to 11 pigs each. Pigs in group 1 served as unchallenged controls. Pigs in groups 2 to 7 were challenged intranasally with 2 ml of high-virulence PRRSV isolate VR-2385 (10
4.47
50% tissue culture infective doses per 2 ml) on day 0 of the study (30 days of age). Seven days after PRRSV challenge, pigs in groups 2 to 7 were challenged intranasally with 2 ml of
Streptococcus suis
serotype 2 (10
8.30
CFU/2 ml). Group 2 pigs served as untreated positive controls. Antimicrobial treatments included daily intramuscular injection with 66,000 IU of procaine penicillin G per kg of body weight on days 8 to 10 (group 3), drinking water medication with 23.1 mg of tiamulin per kg during days 8 to 10 (group 4), and daily intramuscular injection of 5.0 mg of ceftiofur hydrochloride per kg on days 8 to 10 (group 5). Vaccination regimens included two intramuscular doses of an autogenous killed
S. suis
vaccine (group 6) prior to
S. suis
challenge or a single 2-ml intramuscular dose of an attenuated live PRRSV vaccine (group 7) 2 weeks prior to PRRSV challenge. Mortality was 0, 63, 45, 54, 9, 40, and 81% in groups 1 to 7, respectively. Ceftiofur treatment was the only regimen that significantly (
P
< 0.05) reduced mortality associated with PRRSV and
S. suis
coinfection. The other treatments and vaccinations were less effective. We conclude that ceftiofur administered by injection for three consecutive days following
S. suis
challenge was the most effective regimen for minimizing disease associated with PRRSV and
S. suis
coinfection.
Molecular Characterization of
Cryptosporidium
Isolates Obtained from Human Immunodeficiency Virus-Infected Individuals Living in Switzerland, Kenya, and the United States
Authors: Morgan Una , Weber Rainer , Xiao Lihua , Sulaiman Irshad , Thompson R. C. Andrew , Ndiritu Wangeci , Lal Altaf , Moore Anne , Deplazes Peter
Published: December 2019

A total of 22
Cryptosporidium
isolates from human immunodeficiency virus-infected patients from Kenya, Switzerland, and the United States were examined at three genetic loci: the 18S ribosomal DNA, HSP-70, and acetyl coenzyme A synthetase genes. Four distinct
Cryptosporidium
genotypes were identified: (i) the
Cryptosporidium parvum
“human” genotype, (ii) the
C. parvum
“cattle” genotype, (iii)
Cryptosporidium felis
, and (iv)
Cryptosporidium meleagridis
. This is the first report of
C. meleagridis
in a human host. These results and those of others indicate that immunocompromised individuals are susceptible to a wide range of
Cryptosporidium
species and genotypes. Future studies are required to understand the full public health significance of
Cryptosporidium
genotypes and species in immunocompromised hosts.
Novel Screening Method for Urine Cultures Using a Filter Paper Dilution System
Authors: Kunin Calvin M. , Buesching William J.
Published: December 2019

We have developed a novel method for urine culture for office practice based on the use of filter paper as a solid-phase dilution device. Filtration dilutes and spreads the inoculum onto a solid culture surface. Experiments were conducted to determine the optimum inoculum size, microbial permeability through filter papers, and ability to exclude vaginal epithelial cells. The filter paper dilution system was compared to the standard streak method to detect bacteriuria in specimens submitted to the diagnostic laboratory. The sensitivity and specificity of the filter paper dilution system for detection of high-count (≥10
4
CFU/ml) gram-negative bacteriuria in 487 urine specimens were 98.2 and 97.4%, respectively. The sensitivity and specificity for gram-positive bacteriuria in 404 urine specimens were 91.2 and 99.2%, respectively. Low-count gram-negative bacteriuria (<10
4
CFU/ml) was detected by the filter paper dilution system in five of nine specimens (55.6%). In addition, the filter paper dilution system was able to detect gram-negative bacteria in 12 of 41 (29.3%) mixed cultures.
Lactobacillus
and
Gardnerella
organisms in urine specimens were excluded by the filter paper dilution system. Only three of eight
Candida
sp. isolates were detected at counts of ≥10
4
CFU/ml. The system has good storage properties and can be inoculated at the point of source without the need for refrigeration or preservatives. It should be a useful screening method for office practice, where members of the family
Enterobacteriaceae
and staphylococci cause most infections. Standard culture methods are preferred for hospital diagnostic microbiology laboratories, where there is a need to detect yeasts and fastidious microorganisms and to isolate individual colonies from mixed cultures.
Otitis Externa Associated with
Malassezia sympodialis
in Two Cats
Authors: Crespo M. J. , Abarca M. L. , Cabañes F. J.
Published: December 2019

The lipid-dependent species
Malassezia sympodialis
was isolated from two cats with otitis externa. To our knowledge, this is the first report of the isolation of lipid-dependent species of the genus
Malassezia
associated with skin disease in domestic animals.
Outbreak of Infections Caused by
Pseudomonas aeruginosa
Producing VIM-1 Carbapenemase in Greece
Authors: Tsakris Athanassios , Pournaras Spyros , Woodford Neil , Palepou Marie-France I. , Babini Gioia S. , Douboyas John , Livermore David M.
Published: December 2019

Resistance to imipenem and meropenem was observed in 211 (16.5%) isolates of
Pseudomonas aeruginosa
recovered in a Greek university hospital during 1996 to 1998. In six isolates selected from throughout this period, high-level resistance to both carbapenems (MICs ≥ 128 μg/ml) was associated with production of the class B β-lactamase VIM-1.
bla
VIM
-bearing isolates belonged to serotype O:12 and were indistinguishable by pulsed-field gel electrophoresis.
Serological and Virological Characterization of Clinically Diagnosed Cases of Measles in Suburban Khartoum
Authors: Mubarak H. Sittana El , Van De Bildt Marco W. G. , Mustafa Omer A. , Vos Helma W. , Mukhtar Maowia M. , Groen Jan , Hassan Ahmed M. El , Niesters Hubert G. M. , Ibrahim Salah A. , Zijlstra Edward E. , Wild T. Fabian , Osterhaus Albert D. M. E. , De Swart Rik L.
Published: December 2019

Measles continues to be a major childhood disease in terms of global morbidity and mortality. In the main areas of its endemicity the only available means of diagnosis are based on clinical criteria: the presence of a maculopapular rash and fever accompanied by cough, coryza, and/or conjunctivitis. We have studied 38 clinically diagnosed cases of measles in Khartoum, Sudan, by means of serology, reverse transcriptase PCR (RT-PCR) on throat swabs and virus isolation from lymphocytes. On the basis of serology, 28 patients were diagnosed as having an acute measles virus (MV) infection, while in 10 cases the clinical symptoms proved to have other causes. It was shown that in cases with low serum immunoglobulin M (IgM) levels, an additional measurement of IgG or virus-neutralizing antibodies was necessary to discriminate between patients with an acute MV infection sampled during an early stage of the disease and patients who had experienced an MV infection in the more distant past. The serological laboratory diagnosis was validated by an MV-specific RT-PCR: for all confirmed measles cases tested a fragment of the correct size which hybridized with a third MV-specific primer could be amplified, while all serologically negative cases were also RT-PCR negative. MV could be isolated from 17 out of 23 of the serologically confirmed cases, demonstrating that virus isolation is less reliable as a diagnostic tool than serology or RT-PCR. This study stresses the urgent need for a rapid diagnostic field test for measles.
Emergence of Drug Resistance Mutations in Human Immunodeficiency Virus Type 2-Infected Subjects Undergoing Antiretroviral Therapy
Authors: Rodés Berta , Holguín Africa , Soriano Vincent , Dourana Manuela , Mansinho Kamal , Antunes Francisco , González-Lahoz Juan
Published: December 2019

The reverse transcriptase (RT) and protease genes from 12 human immunodeficiency virus type 2 (HIV-2)-infected individuals who had been exposed to antiretroviral drugs for longer than 6 months were examined for the presence of mutations which could be involved in drug resistance. Four individuals carried virus genotypes with amino acid substitutions potentially associated with resistance to nucleoside analogues: two at codon 70 (K→R) and two at codon 184 (M→V). Moreover, the latter two patients harbored a codon Q151M mutation which is associated to multidrug resistance in HIV-1, and one of these subjects carried some of the typically linked mutations at codons 65 and 69. With regard to the protease inhibitors, substitutions associated with resistance to protease inhibitors at codon 46 were observed in all individuals. Moreover, minor resistance mutations, as well as new ones of unknown meaning, were often seen in the protease gene. In conclusion, amino acid changes in the HIV-2 RT and protease genes which could be associated with drug resistance seem to occur at positions identical to those for HIV-1.