bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of MICs of ceftiofur and other antimicrobial agents against bacterial pathogens of swine from the United States, Canada, and Denmark
Authors: Salmon S A , Watts J L , Case C A , Hoffman L J , Wegener H C , Yancey R J
Published: January 2020

The MICs of ceftiofur and other antimicrobial agents, tested for comparison, for 515 bacterial isolates of pigs from the United States, Canada, and Denmark with various diseases were compared. The organisms tested included Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus suis, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi subsp. equi, and Streptococcus equi subsp. zooepidemicus. In addition to ceftiofur, the following antimicrobial agents or combinations were tested: enrofloxacin, ampicillin, sulfamethazine, trimethoprim-sulfadiazine (1:19), erythromycin, lincomycin, spectinomycin, lincomycin-spectinomycin (1:8), tilmicosin, and tetracycline. Tilmicosin was only tested against the U.S. isolates. Overall, ceftiofur and enrofloxacin were the most active antimicrobial agents tested against all isolates, with MICs inhibiting 90% of isolates tested (MIC90s) of < or = 2.0 and < or = 1.0 microgram/ml, respectively. Erythromycin, sulfamethazine, spectinomycin, and lincomycin demonstrated limited activity against all of the organisms tested, with MIC90s of > or = 8.0, > or = 256.0, > or = 32.0, and > or = 16.0 micrograms/ml, respectively. Trimethoprim-sulfadiazine was active against isolates of A. pleuropneumoniae, S. choleraesuis, S. typhimurium, P. multocida, S. equi, and S. suis (MIC90s, < or = 0.5 microgram/ml) but was less active against the E. coli strains tested (MIC90, > 16.0 micrograms/ml). Ampicillin was active against the P. multocida, S. suis, and S. equi isolates tested (MIC90s, 0.5, 0.06, and 0.06 micrograms/ml, respectively) and was moderately active against S. typhimurium (MIC90s, 2.0 micrograms/ml). However, this antimicrobial agent was much less active when it was tested against A. pleuropneumoniae, S. cholerae-suis, and E. coli (MIC90s, 16.0, > 32.0, and 32.0 micrograms/ml, respectively). Against the U.S. isolates of A. pleuropneumoniae and P. multocida, tilmicosin was moderately active (MIC90s, 4.0 and 8.0 micrograms/ml, respectively). However, this compound was not active against the remaining U.S. isolates (MIC90s, > 64.0 micrograms/ml). Differences in the MICs from one country to another were not detected with enrofloxacin, ceftiofur, or lincomycin for the strains tested, but variations in the MICs of the remaining antimicrobial agents were observed.
A simple and sensitive DNA hybridization assay used for the routine diagnosis of human parvovirus B19 infection
Authors: Hicks K E , Beard S , Cohen B J , Clewley J P
Published: January 2020

A dot blot hybridization assay for parvovirus B19 diagnosis was developed by using a PCR-generated probe, digoxigenin labelling, and chemiluminescence detection. Different labelling techniques and hybridization solutions were evaluated. From this analysis a protocol was devised for routine diagnostic use. The protocol enabled 1 pg of B19 DNA to be detected. The results of applying this method to 8,369 diagnostic samples collected during 1994 and 1995 are given.
Catheter-related bacteremia associated with coagulase-positive Staphylococcus intermedius
Authors: Vandenesch F , Célard M , Arpin D , Bes M , Greenland T , Etienne J
Published: January 2020

We report a case of catheter-related bacteremia in a 63-year-old patient caused by Staphylococcus intermedius. Clinical resolution of the infection was obtained after removal of the intravenous device and antibiotic treatment. This observation emphasizes the risk of confusion between S. intermedius and Staphylococcus aureus if only a coagulase test is done.
Comparative evaluation of BACTEC aerobic Plus/F and Septi-Chek Release blood culture media
Authors: Rohner P , Pepey B , Auckenthaler R
Published: January 2020

The BACTEC 9240 (Becton Dickinson, Sparks, Md.) automated blood culture system is based on the continuous monitoring of CO2 production by means of a fluorescent sensor attached to the bottom of culture vials. We compared the performance of the BACTEC aerobic Plus/F medium to that of the Septi-Chek Release medium (Becton Dickinson, Sparks, Md.), a manual biphasic blood culture system. Sets consisting of BACTEC aerobic Plus/F and Septi-Chek Release vials inoculated with similar volumes (7 +/- 2 ml) were evaluated. In the laboratory, systems were equipped and analyzed according to the manufacturer's recommendations. The BACTEC and Septi-Chek vials were incubated at 35 degrees C for 5 days. A total of 6,116 compliant sets were obtained from 1,972 adult patients (3.1 cultures per patient). Of these, 731 (12%) were culture positive, including 612 (10%) that yielded at least one pathogen, and 143 (2%) were considered to be contaminated. Of the 672 pathogenic organisms detected, 524 were isolated from the BACTEC aerobic Plus/F medium and 574 were isolated from the Septi-Chek Release medium, 428 organisms grew in both media, 96 organisms grew only in the BACTEC aerobic Plus/F medium, and 146 organisms grew only in the Septi-Chek Release medium (P = 0.001). Haemophilus spp. were isolated more often (P = 0.03) from the BACTEC aerobic Plus/F medium; however, more Streptococcus anginosus organisms (P = 0.02), members of the Enterobacteriaceae family (P < 0.03), and gram-negative anaerobes (P = 0.03) were isolated from the Septi-Chek Release medium. Pathogenic organisms were detected significantly earlier (P < 0.0001) with the BACTEC aerobic Plus/F medium in conjunction with the BACTEC 9240 instrument than with the Septi-Chek Release medium.
Field inversion gel electrophoretic analysis of Legionella pneumophila strains associated with nosocomial legionellosis in children
Authors: Green M , Wald E R , Dashefsky B , Barbadora K , Wadowsky R M
Published: January 2020

Two nosocomial cases of Legionnaires' disease occurred in children. Legionella pneumophila serogroup 1 was isolated from both patients and 30 of 39 plumbing system sites in the hospital. The patient and hospital environmental isolates yielded identical field inversion gel electrophoretic patterns which differed from patterns observed with epidemiologically unrelated strains.
Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive care unit and dissemination of the extended-spectrum beta-lactamase to other members of the family enterobacteriaceae
Authors: Neuwirth C , Siebor E , Lopez J , Pechinot A , Kazmierczak A
Published: January 2020

We report an outbreak of Enterobacter aerogenes in an intensive care unit (ICU) and two medicine departments that produced the extended-spectrum beta-lactamase TEM-24, which was difficult to detect by disk agar diffusion. The strains were compared by DNA restriction fragment length polymorphism after pulsed-field gel electrophoresis following cleavage with XbaI. This typing method indicated that a single strain, first isolated in the ICU, spread throughout the other medical departments as a result of patient transfer. We also observed the transfer in vivo of the plasmid encoding TEM-24 from the strain of Enterobacter aerogenes to different strains of Escherichia coli and Citrobacter freundii in the ICU. It therefore appears that the epidemic involved results from two events: dissemination of one strain of Enterobacter aerogenes and dissemination of the plasmid encoding TEM-24 among various members of the family Enterobacteriaceae.
Monoclonal infection involving Mycobacterium avium presenting with three distinct colony morphotypes
Authors: Wright E L , Zywno-van Ginkel S , Rastogi N , Barrow W W
Published: January 2020

Recent reports indicate that polyclonal infections may play an important role in multiple drug resistance in Mycobacterium avium infections. We report here on the isolation of a single M. avium strain that appeared to have smooth colony morphology upon initial isolation on a Lowenstein-Jensen slant. Primary subculture onto Middlebrook 7H10, however, revealed three distinct morphotypes representing smooth opaque (SmO), smooth transparent (SmT), and rough (Rg) colony morphologies. All three morphotypes were identified as M. avium by standard biochemical procedures, Genprobe analysis, and mycolic acid patterns. Subsequent restriction fragment length polymorphism analysis, using SalI- and PvuII-digested genomic DNA, revealed identical patterns for hybridization with the IS1245 probe. Thin-layer chromatographic analysis of lipids from the three morphotypes revealed that only the SmT morphotype possessed what appeared to be lipid components similar to, but unlike, previously described serovar-specific glycopeptidolipid antigens. Further analysis of internally radiolabeled deacylated lipids from the SmT morphotype, by high-performance liquid chromatography and thin-layer chromatography, disclosed that some of these components can be internally radiolabeled with [14C] phenylalanine and [14C]mannose. These results suggest that these components are structurally similar to previously described glycopeptidolipid antigens. This is apparently the first report of a monoclonal infection involving a single strain of M. avium presenting with all three colony morphotypes, SmO, SmT, and Rg.
Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis
Authors: Millar M R , Linton C J , Cade A , Glancy D , Hall M , Jalal H
Published: January 2020

The purpose of the present study was to determine the extent to which bacteria not detected by culture contribute to the microbial flora of the bowel of preterm infants with and without neonatal necrotizing enterocolitis (NEC). Fecal samples from 32 preterm infants in special care baby units including samples from 10 infants with NEC were examined by culture and PCR amplification of the 16S rRNA gene (rDNA). The 16S rDNA V3 region was amplified with eubacterial primers, and the amplification products derived from the fecal sample DNA were compared with the products from individual cultured isolates by PCR and denaturing gradient gel electrophoresis (PCR-DGGE), allowing the DNA from uncultured bacteria to be identified. For the 22 infants without NEC weekly samples were examined for a mean of 5.3 postnatal weeks. The total number of types detected by culture combined with PCR-DGGE was 10.1 per infant, of which PCR-DGGE contributed 10.4% of the types identified. Additional types detected by PCR-DGGE were found in 14 (63.6%) of the 22 infants. The majority of the sequences associated with uncultured bacteria showed > 90% 16S rDNA sequence identity with sequences from culturable human enteric flora, and all were found in single infants with the exception of sequences indistinguishable by DGGE from seven infants. These sequences showed > 90% sequence identity with the 16S rDNA of Streptococcus salivarius and may have been derived from upper gastrointestinal or respiratory tract flora. In the present study uncultured bacteria detected by PCR-DGGE were no more frequent in fecal samples from infants with NEC than in samples from infants without NEC, although these findings do not exclude the possibility of unrecognized bacteria associated with the mucosa of the small intestine of infants with NEC.
Identification of Turicella otitidis isolated from a patient with otorrhea associated with surgery: differentiation from Corynebacterium afermentans and Corynebacterium auris
Authors: Renaud F N , Grégory A , Barreau C , Aubel D , Freney J
Published: January 2020

Turicella otitidis is a newly described coryneform bacterium isolated from middle ear fluids. We report here on the diagnosis of a strain isolated from otorrhea. The API Coryne system (bioMérieux, Marcy I'Etoile, France) used alone failed to differentiate T. otitidis, Corynebacterium afermentans, and Corynebacterium auris (ANF group). Biochemical tests such as DNase, enzymatic reactions (API ZYM; bioMérieux), and carbon substrate assimilation tests (Biotype 100; bioMérieux) allow presumptive identification. However, only chemotaxonomy and molecular biology can achieve unequivocal differentiation among these three species.
Prevalence of human herpesvirus 8 DNA sequences in several patient populations
Authors: Marchioli C C , Love J L , Abbott L Z , Huang Y Q , Remick S C , Surtento-Reodica N , Hutchison R E , Mildvan D , Friedman-Kien A E , Poiesz B J
Published: January 2020

We have undertaken a large-scale study of various tissues from normal controls and patients with Kaposi's sarcoma (KS) or other malignancies, both with and without human immunodeficiency virus infection, to determine the prevalence of human herpesvirus 8 (HHV-8) DNA. A total of 566 specimens were analyzed by PCR for the presence of HHV-8 DNA. Of the samples tested, 251 were obtained from patients with KS and 315 were obtained from patients without KS. HHV-8 DNA was detected in 103 (41%) of the 251 samples from patients with KS. In particular, 92% of KS tumor specimens were positive. None of the tissues from patients without KS showed evidence of HHV-8 DNA. Sequencing and phylogenetic analyses indicate a high degree of conservation (97.5 to 100%) among the HHV-8 strains tested.
Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, GeneReleaser, and the phenol-chloroform method
Authors: Kramvis A , Bukofzer S , Kew M C
Published: January 2020

The abilities of GeneReleaser and QIAamp to extract the hepatitis B virus (HBV) DNA template from serum for amplification by PCR were evaluated and compared with that of the standard phenol-chloroform method. Differences in the sensitivities of the three methods were revealed by nested PCR of HBV DNA extracted from serially diluted hepatitis B e antigen (HBeAg)-positive (high-titer) serum. Phenol-chloroform was found to be the most sensitive extraction method but was time-consuming and labor intensive, and the many steps required increased the possibility of contamination. In a titration of HBeAg-negative (low-titer) serum, all three methods coupled with nested PCR were capable of detecting low levels of HBV DNA. In the case of QIAamp and GeneReleaser, the extraction was relatively simple and rapid. The higher quantity of serum (200 microliters) used in the QIAamp extraction did not provide higher sensitivity, possibly because of incomplete removal of Taq polymerase inhibitors from the serum or inadequate disruption of the virion. GeneReleaser was more efficient because it gave the same detection limit in low-titer serum as phenol-chloroform even though it utilizes only 5 microliters of serum. However, it did not produce consistent amplifications of HBV DNA, giving false-negative results in 7 of the 50 cases (14%) in one experiment. Use of a larger volume of serum and replicate extractions may overcome this problem. Advantages thus exist in each of the extraction methods, and these should be weighed against the disadvantages when deciding which extraction method is appropriate.
On the nature and use of randomly amplified DNA from Staphylococcus aureus
Authors: van Leeuwen W , Sijmons M , Sluijs J , Verbrugh H , van Belkum A
Published: January 2020

Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing Eco RI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n = 12), hybridized in a homogeneously positive fashion (n = 13) or hybridized only with their source strains (n = 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.
Rapid detection and isolation of shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay
Authors: Beutin L , Zimmermann S , Gleier K
Published: January 2020

By combining the enterohemolysin test and the VTEC-RPLA test (specific for the detection of Shiga-like toxin I [SLT-I], SLT-II, and SLT-IIc), single colonies of SLT-producing Escherichia coli were found to constitute between 0.03 and 68.1% of the coliform flora in human stool cultures and were isolated and characterized within 72 to 96 h.
Hepatitis C virus genotypes in northern Italy: clinical and virological features
Authors: Ravaggi A , Rossini A , Mazza C , Puoti M , Marin M G , Cariani E
Published: January 2020

We analyzed the characteristics of subjects from the same area who were infected with hepatitis C virus genotypes 1 through 4 and subtypes 1a and 1b. Our data are consistent with a rapid evolution in the epidemiology of HCV genotypes and argue against different pathogenic potentials for genotypes 1b and 2.
Detection of Cryptococcus neoformans gene in patients with pulmonary cryptococcosis
Authors: Tanaka K , Miyazaki T , Maesaki S , Mitsutake K , Kakeya H , Yamamoto Y , Yanagihara K , Hossain M A , Tashiro T , Kohno S
Published: January 2020

Pulmonary cryptococcosis was diagnosed by nested PCR. Extraction of DNA was performed by mechanical destruction of the capsules of Cryptococcus neoformans by the glass bead technique. Nested PCR was positive for 4 of 5 culture-positive specimens but negative for 1 culture-positive specimen, 10 culture-negative specimens, and 1 specimen with undetermined culture results.
A microsporidian isolated from an AIDS patient corresponds to Encephalitozoon cuniculi III, originally isolated from domestic dogs
Authors: Didier E S , Visvesvara G S , Baker M D , Rogers L B , Bertucci D C , De Groote M A , Vossbrinck C R
Published: January 2020

The ribosomal DNA internal transcribed spacer (ITS) region of a recently cultured human Encephalitozoon cuniculi isolate was analyzed by gene amplification and DNA sequencing. Restriction endonuclease digestion (FokI) and double-stranded DNA heteroduplex mobility shift analysis were performed to determine their utility for strain differentiation. The human E. cuniculi isolate was identical to E. cuniculi III, which had been isolated only from domestic dogs until now. The patient providing the isolate owned a pet dog, but no microsporidia were detected in the pet's urine.
Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay
Authors: Müller N , Zimmermann V , Hentrich B , Gottstein B
Published: January 2020

A recently described PCR test for the identification of Neospora caninum and Toxoplasma gondii has been further developed and optimized in view of its practicability for routine diagnostic application. The N. caninum-specific PCR was adapted to the diagnostic operating standard of the T. gondii-specific PCR in that the uracil DNA glycosidase system was introduced, which eliminates potential carry-over contaminations of amplified target DNA from previous reactions. Furthermore, both PCR tests were optimized by including a DNA hybridization immunoassay based on the use of the commercially available Gen-eti-k DEIA kit. This assay allowed highly sensitive and specific detection of respective DNA amplification products and thus substantially facilitated the reading and interpretation of the test results.
Clonal and temporal patterns of nasopharyngeal penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae stains in children attending a day care center
Authors: Pons J L , Mandement M N , Martin E , Lemort C , Nouvellon M , Mallet E , Lemeland J F
Published: January 2020

The nasopharyngeal carriage of penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae (PSSp and PRSp, respectively) was analyzed in 116 children attending a day care center in Rouen, France, by three observation periods in November, January, and March of the winter of 1993 to 1994. The carriage rate of S. pneumoniae was found to be 47.7, 47.3, and 49.6% at each different observation period, and PRSp accounted for 42.2, 40.3, and 40.6% of pneumococcal isolates, respectively. The 52 isolates recovered in November were distributed in 34 electrophoretic types (ETs) by multilocus enzyme electrophoresis; 15 PRSp isolates, all of serotype 23F, belonged to a clonal complex of five ETs, representing the dominant population of PRSp in November. The temporal pattern of S. pneumoniae carriage was studied in 17 children who were colonized at the three periods by multilocus enzyme analysis of their isolates. The PSSp isolated, exhibiting distinct ETs, were transient only among these day care attendees. In contrast, most of the PRSp isolated in January and March belonged to the clonal complex. Thus, this PRSp population was resident in the day care center throughout the study period.
Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O:12 outbreak isolates from a pediatric hospital
Authors: Bingen E , Bonacorsi S , Rohrlich P , Duval M , Lhopital S , Brahimi N , Vilmer E , Goering R V
Published: January 2020

Ribotyping randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis were used for the epidemiologic evaluation of eight Pseudomonas aeruginosa O:12 isolates obtained from eight children and two P. aeruginosa O:12 environmental isolates from a hematology ward. Randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis were able to discriminate isolates that were indistinguishable by biochemical typing, O serotyping or ribotyping.
Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study
Authors: Kirschner P , Rosenau J , Springer B , Teschner K , Feldmann K , Böttger E C
Published: January 2020

We have investigated the use of DNA amplification by PCR for the detection of mycobacteria in clinical specimens, with the gene encoding the 16S rRNA as a target. Following generic amplification of mycobacterial nucleic acids, screening was done with genus-specific probe; this was followed by species differentiation by use of highly discriminating probes or nucleic acid sequencing. In a prospective 18-month evaluation, criteria to select specimens for PCR analysis were defined. Of a total of 8,272 specimens received, 729 samples satisfied the criteria and were subjected to DNA amplification. Clinical specimens included material from the respiratory tract (sputa and bronchial washings), aspirates, biopsies, and various body fluids (cerebrospinal, pleural, peritoneal, and gastric fluids). After resolution of discrepant results, the sensitivity of the PCR assay was 84.5%, the specificity was 99.5%, the positive predictive value was 97.6%, and the negative predictive value was 96.4%. The sensitivity and negative predictive value of culture (with a combination of broth and solid media) were 77.5 and 94.8%, respectively. In conclusion, this PCR assay provides an efficient strategy to detect and identify multiple mycobacterial species and performs well in comparison with culture.
Typing multidrug-resistant Staphylococcus aureus: conflicting epidemiological data produced by genotypic and phenotypic methods clarified by phylogenetic analysis
Authors: Jorgensen M , Givney R , Pegler M , Vickery A , Funnell G
Published: January 2020

An outbreak of an unusual tetracycline-sensitive, rifampicin- and ciprofloxacin-resistant, methicillin-resistant Staphylococcus aureus (MRSA) strain at a large teaching hospital was investigated. Two typing methods, phage typing and restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (RFLP-PFGE), gave conflicting results which were clarified by phylogenetic analysis. Phage typing identified all the "epidemic-associated" strains as identical, while RFLP-PFGE further divided these strains into four pulsotypes. Phylogenetic analysis showed these four pulsotypes were related genetically and also recognized a second strain of MRSA causing a continuing cross-infection problem. Variation in the RFLP-PFGE pattern was shown to occur following lysogenization of phage-sensitive MRSA. These results indicate that in analyzing outbreaks caused by subgroups of clonal organisms like MRSA, it is necessary to use at least two typing methods and that conflicts between these could be resolved by phylogenetic analysis.
Effect of antibiotics contained in two Brucella selective media on growth of Brucella abortus, B. melitensis, and B. ovis
Authors: Marín C M , Alabart J L , Blasco J M
Published: January 2020

The MIC and the highest concentration enabling bacterial growth (CEG) of the antibiotics contained in two selective media were determined for Brucella abortus, B. melitensis, and B. ovis. The nalidixic acid and bacitracin contained in Farrell's selective medium were responsible for the inhibitory effects observed.
Expression of recombinant parvovirus NS1 protein by a baculovirus and application to serologic testing of rodents
Authors: Riley L K , Knowles R , Purdy G , Salomé N , Pintel D , Hook R R , Franklin C L , Besch-Williford C L
Published: January 2020

A recombinant baculovirus containing the NS1 gene of minute virus of mice was constructed. Optimal expression of the recombinant NS1 protein (rNS1) was achieved by infecting Trichoplusa ni High Five cells at a multiplicity of 10 and incubating them for 72 h postinfection. An enzyme-linked immunosorbent assay (ELISA) with rNS1 as the antigen was evaluated for serologic testing of laboratory rodents. The rNS1 ELISA proved to be a more sensitive method for the detection of antibodies to recently recognized rodent parvovirus species (mouse orphan parvovirus and rat orphan parvovirus) and prototypic parvovirus species (minute virus of mice, Kilham's rat virus, and H-1) than were conventional parvovirus ELISAs that use whole parvovirus virions.
Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR
Authors: Folgueira L , Delgado R , Palenque E , Aguado J M , Noriega A R
Published: January 2020

A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.
Quantitative light microscopic detection of Enterocytozoon bieneusi in stool specimens: a longitudinal study of human immunodeficiency virus-infected microsporidiosis patients
Authors: Clarridge J E , Karkhanis S , Rabeneck L , Marino B , Foote L W
Published: January 2020

The clinical course of microsporidiosis caused by Enterocytozoon bieneusi and the pattern of intestinal shedding of spores have not been correlated, at least in part because detection of E. bieneusi in stools is more difficult than detection of other protozoa because of its smaller size and less intense staining. We examined with a modified trichrome stain 124 stool specimens collected over a 2-year follow-up period from 23 human immunodeficiency virus-infected patients with electron microscopic-proven E. bieneusi infection and correlated the results with electron microscopic observations from duodenal biopsy specimens taken at the beginning of the study period. E. bieneusi was detected in the stool at least once in 74% (17 of 23) of all patients, in 100% (9 of 9) of patients in whose tissue moderate or abundant numbers of parasites were seen, and in 57% (8 of 14) of patients in whose tissue few parasites were seen. In two patients with abundant tissue parasites, many microsporidia were detected in every stool specimen (13 of 13) during the follow-up period, whereas among the patients with few tissue parasites, only 23% (15 of 64) of stool specimens were positive. Furthermore, if spore stages as well as plasmodial stages were detected in tissue, stool specimens were more likely to be positive. Although most of the heavily infected stools were from patients with chronic diarrhea, microsporidia were detected in 33, 28, and 42% of stool specimens from patients with nil, intermittent, and chronic diarrhea patterns, respectively. Although quantitation of E. bieneusi spores in stool specimens was closely correlated with quantitation in tissue, it was not correlated with reported patterns of diarrhea.
Comparison of Bactec 9240 and Difco ESP blood culture systems for detection of organisms from vials whose entry was delayed
Authors: Chapin K , Lauderdale T L
Published: January 2020

A comparison of the Bactec 9240 (Becton-Dickinson, Sparks, Md.) and Difco ESP (Difco Laboratories, Detroit, Mich.) instruments for the detection of organism growth from vials whose entry was delayed was evaluated. The instruments' capabilities for organism recovery, time to detection, rates of false-positive results, and numbers of vials in which growth was not detected were made by using seeded blood culture vial pairs and controls with and without delayed entry. Bactec 9240 and Difco ESP aerobic and anaerobic vials were inoculated with human blood and were seeded with organism growth from 18 species, including obligate aerobic, anaerobic, and facultative anaerobic organisms. Each organism was tested in triplicate at 0, 8, 24, 36, and 48 h and was incubated at both room temperature (RT) and 35 degrees C. Two separate phases of the study were performed, each with a different version of Bactec 9240 software. Overall, detection of growth in vials with delayed entry into either the Bactec 9240 or the Difco ESP instrument resulted in an increased total time to detection with incubation at both RT and 35 degrees C compared with the total time to detection for nondelayed vials. However, false-positive results and vials in which growth was not detected were minimal, and delayed entry did not require routine entry or exit subcultures for either system. Analysis of individual time points and incubation temperatures for the detection of all organisms suggested that Difco ESP vials delayed by up to 8 h may be incubated at 35 degrees C (100% detection) and vials delayed for longer than 8 h may remain at RT. Bactec 9240 vials may be incubated at 35 degrees C for up to 24 h with a minimal loss of detection (97.9% detection), and vials delayed for more than 24 h should remain at RT for optimal recovery of organism growth.
Molecular characteristics of Haemophilus influenzae causing invasive disease during the period of vaccination in Switzerland: analysis of strains isolated between 1986 and 1993
Authors: Muhlemann K , Balz M , Aebi S , Schopfer K
Published: January 2020

The broad use of conjugated vaccines against Haemophilus influenzae type b may select for strains to which the polysaccharide vaccine does not provide immunity. We analyzed 392 consecutive H. influenzae isolates from Swiss children 0 to 16 years of age with invasive disease during the years 1986 to 1993. Bacterial strains were characterized by serotyping, capsular genotyping, outer membrane protein (OMP) subtyping, and ribotyping. Of 392 strains, 372 were serotype b, 1 was serotype a, 3 were serotype f, and 16 were nontypeable H. influenzae. After the introduction of Haemophilus conjugate vaccines in 1990, there was a relative increase of nontypeable strains from 3 to 6.6% (P = 0.27). Of the type b strains, 281 (75.5%) had the same OMP subtype and ribotype pattern. This clone predominated in the pre- and postvaccine periods. After the year 1990, the proportions of OMP subtype 1c and OMP subtype 3 tended to increase. Isolates from previously vaccinated (n = 10) and nonvaccinated patients did not differ in their subtype distributions. We conclude that the administration of conjugated vaccines decreased invasive disease caused by the most prevalent H. influenzae type b clone. However, further surveillance of circulating H. influenzae strains during the period of vaccination is indicated.
Detection of Burkholderia pseudomallei DNA in patients with septicemic melioidosis
Authors: Dharakul T , Songsivilai S , Viriyachitra S , Luangwedchakarn V , Tassaneetritap B , Chaowagul W
Published: January 2020

Septicemic melioidosis is the most severe form of melioidosis, which is caused by Burkholderia pseudomallei. It is endemic in Southeast Asia and is the leading cause of death from community-acquired septicemia in northeast Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. More than half of the patients die within the first 2 days after hospital admission, before bacterial cultures become positive. The present study was undertaken to develop a rapid diagnostic method for identification of this organism. A nested PCR system that amplified a part of 16S rRNA gene that was highly specific to B. pseudomallei was developed. This system was able to detect as few as two bacteria present in the PCR. DNAs from all 30 clinical isolates of B. pseudomallei and none of the other bacteria tested were amplified. The described PCR system has been employed for the detection of the organism in clinical specimens, including buffy coat and pus from internal organs. The detection of B. pseudomallei in buffy coat specimens by PCR was shown to be comparable to the detection of bacteria from blood cultures in septicemic melioidosis cases.
Multicenter evaluation of new double-antigen sandwich enzyme immunoassay for measurement of anti-human immunodeficiency virus type 1 and type 2 antibodies
Authors: Burgisser P , Simon F , Wernli M , Wust T , Beya M F , Frei P C
Published: January 2020

A new enzyme immunoassay (EIA), the Cobas Core Anti-HIV-1/HIV-2 EIA DAGS (also referred to as Roche DAGS), for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 was evaluated in four centers. The assay is based on the double-antigen sandwich (DAGS) format, which enables the detection of all classes of antibodies. The antigens consist of recombinant proteins in their native conformation and of synthetic peptides. Of a total of 5,836 negative serum samples, including 95 samples likely to produce false reactivities, 6 were false positive, resulting in a specificity of 99.9%. None of 35 sera that were from noninfected individuals but contained p24-cross-reacting antibodies as revealed by Western blot (immunoblot) analysis were reactive by the Roche DAGS assay. In samples from individuals infected with HIV-1 group M (n = 499) and HIV-2 (n = 200), the sensitivity of the assay was 100%. Although containing antigens with sequences from subtype B only, the assay was also able to correctly identify with high optical density/cutoff ratios samples from subjects infected with HIV-1 subtype O (n = 10). In 17 of 19 seroconversion panels tested, the assay detected the presence of HIV-1 antibodies as early as another sandwich EIA. Eight of these panels were also analyzed by an indirect second-generation assay, which detected antibodies 2 to 10 days later than did the DAGS assay under evaluation. The excellent specificity and sensitivity of the new Cobas Core Anti-HIV-1/HIV-2 EIA DAGS are the result of the DAGS format as well as of the native, naturally folded form of the recombinant protein used as the gag antigen.
Moredun Bordetella Medium, an improved selective medium for isolation of Bordetella parapertussis
Authors: Connor K M , Porter J F , Quirie M M , Donachie W
Published: January 2020

Bordetella parapertussis, previously thought to be an obligate human respiratory tract pathogen, has been isolated from sheep. Attempts to assess the prevalence of B. parapertussis in conventionally reared sheep by nasal swabbing proved futile with existing selective media because of extensive overgrowth with Mucor spp. and other nasal commensals. Moredun Bordetella Medium (MBM), which contains cycloheximide and spectinomycin at final concentrations of 0.5 mg/ml and 100 mu g/ml, respectively, was developed as an improved selective medium to isolate B. parapertussis from the nasal cavities of conventionally reared sheep. The selective ability of MBM was evaluated with 200 nasal swabs from conventionally reared sheep, and B. parapertussis was recovered from 31.5% of the samples. MBM facilitated the simple and effective isolation of B. parapertussis from ovine nasal swabs and, in successfully excluding overgrowth with other contaminants, proved superior to other test formulations evaluated and to existing conventional media.
Q fever pneumonia in children in Japan
Authors: To H , Kako N , Zhang G Q , Otsuka H , Ogawa M , Ochiai O , Nguyen S V , Yamaguchi T , Fukushi H , Nagaoka N , Akiyama M , Amano K , Hirai K
Published: January 2020

The prevalence of Q fever pneumonia among children with atypical pneumonia from whom only an acute-phase serum sample was available was traced by using an indirect immunofluorescence (IF) test, nested PCR, and isolation. Twenty (34.5%) of 58 sera were found to have both polyvalent and immunoglobulin M antibodies to the phase II antigen of Coxiella burnetii by the IF test. Q fever pneumonia was present in 23 (39.7%) of 58 patients as determined by both the nested PCR and isolation and in 20 patients as determined by the IF test. The sensitivities for nested PCR and isolation were 100%, and that for the IF test was 87%. Our results indicate that nested PCR was faster and more sensitive than isolation and the IF test in the diagnosis of acute Q fever when a single acute-phase serum was available. These findings suggest that C. burnetii is an important cause of atypical pneumonia in children in Japan.
Immune response to hepatitis A virus capsid proteins after infection
Authors: Wang C H , Tschen S Y , Heinricy U , Weber M , Flehmig B
Published: January 2020

This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.
Rapid differentiation of Mycobacterium avium and M. paratuberculosis by PCR and restriction enzyme analysis
Authors: Eriks I S , Munck K T , Besser T E , Cantor G H , Kapur V
Published: January 2020

Mycobacterium avium subsp. avium (M. avium) and M. avium subsp. paratuberculosis (M. paratuberculosis), intracellular bacteria that can cause chronic granulomatous enteritis in cattle, are difficult to distinguish on the basis of growth and biochemical characteristics. We report the development of a PCR-based strategy for the rapid differentiation of isolates of M. avium from isolates of M. paratuberculosis. Restriction fragment length polymorphism was identified by PCR amplification and subsequent restriction enzyme digestion with PstI of a 960-bp fragment of the 65-kDa heat shock protein (hsp65) from 21 clinical isolates of M. paratuberculosis and 14 isolates of M. avium. These results indicate that a restriction fragment length polymorphism in the hsp65 gene can be used for the rapid differentiation of clinical isolates of M. paratuberculosis and M. avium.
Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas
Authors: Baay M F , Quint W G , Koudstaal J , Hollema H , Duk J M , Burger M P , Stolz E , Herbrink P
Published: January 2020

We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined, 72.8%). The efficacy of each primer pair seemed to be inversely correlated to the length of the amplimer produced. By using newly developed type-specific primer pairs (amplimer length, approximately 100 bp), an increase in HPV DNA detection (87.6%) was found.
HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci
Authors: Goh S H , Potter S , Wood J O , Hemmingsen S M , Reynolds R P , Chow A W
Published: January 2020

A set of universal degenerate primers which amplified, by PCR, a 600-bp oligomer encoding a portion of the 60-kDa heat shock protein (HSP60) of both Staphylococcus aureus and Staphylococcus epidermidis were developed. However, when used as a DNA probe, the 600-bp PCR product generated from S. epidermidis failed to cross-hybridize under high-stringency conditions with the genomic DNA of S. aureus and vice versa. To investigate whether species-specific sequences might exist within the highly conserved HSP60 genes among different staphylococci, digoxigenin-labelled HSP60 probes generated by the degenerate HSP60 primers were prepared from the six most commonly isolated Staphylococcus species (S. aureus 8325-4, S. epidermidis 9759, S. haemolyticus ATCC 29970, S. schleiferi ATCC 43808, S. saprophyticus KL122, and S. lugdunensis CRSN 850412). These probes were used for dot blot hybridization with genomic DNA of 58 reference and clinical isolates of Staphylococcus and non-Staphylococcus species. These six Staphylococcus species HSP60 probes correctly identified the entire set of staphylococcal isolates. The species specificity of these HSP60 probes was further demonstrated by dot blot hybridization with PCR-amplified DNA from mixed cultures of different Staphylococcus species and by the partial DNA sequences of these probes. In addition, sequence homology searches of the NCBI BLAST databases with these partial HSP60 DNA sequences yielded the highest matching scores for both S. epidermidis and S. aureus with the corresponding species-specified probes. Finally, the HSP60 degenerate primers were shown to amplify an anticipated 600-bp PCR product from all 29 Staphylococcus species and from all but 2 of 30 other microbial species, including various gram-positive and gram-negative bacteria, mycobacteria, and fungi. These preliminary data suggest the presence of species-specific sequence variation within the highly conserved HSP60 genes of staphylococci. Further work is required to determine whether these degenerate HSP60 primers may be exploited for species-specific microbic identification and phylogenetic investigation of staphylococci and perhaps other microorganisms in general.
Retrospective survey of chronic Q fever in Japan by using PCR to detect Coxiella burnetii DNA in paraffin-embedded clinical samples
Authors: Yuasa Y , Yoshiie K , Takasaki T , Yoshida H , Oda H
Published: January 2020

We used PCR to detect Coxiella burnetii DNA in paraffin-embedded tissues obtained from patients with chronic endocarditis in which the etiological agent had been unknown. On the basis of the published nucleotide sequence of the C. burnetii htpB gene, primers were chosen to produce an amplified fragment of 285 bp. A total of 60 samples from 56 patients were tested for the presence of C. burnetii DNA. Five samples from four patients were found to be positive. All of the amplified DNA fragments possessed a TthHB8I restriction site, as predicted from the published sequence of C. burnetii. In one of the four positive patients, rickettsia-like particles were found in sections of tissue stained by Gimenez's method. This is the first report of chronic Q fever in Japan.
Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose
Authors: Espinel-Ingroff A , Pfaller M , Erwin M E , Jones R N
Published: January 2020

An interlaboratory evaluation (two centers) of the Etest method was conducted for testing the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Two buffered (phosphate buffer) culture media were evaluated: solidified RPMI 1640 medium with 2% glucose and Casitone agar. MIC endpoints were determined after both 24 and 48 h of incubation at 35 degrees C. Analysis of 3,420 MICs demonstrated higher interlaboratory agreement (percentage of MIC pairs within a 2-dilution range) with Casitone medium than with RPMI 1640 medium when testing amphotericin B (84 to 90% versus 1 to 4%), itraconazole (87% versus 63 to 74%), and ketoconazole (94 to 96% versus 88 to 90%). In contrast, better interlaboratory reproducibility was determined between fluconazole MIC pairs when RPMI 1640 medium rather than Casitone medium was used (96 to 98% versus 77 to 90%). Comparison of the flucytosine MICs obtained with RPMI 1640 medium revealed greater than 80% reproducibility. The study suggests the potential value of the Etest as a convenient alternative method for testing the susceptibilities of yeasts. It also indicates the need for further optimization of medium formulations and MIC endpoint criteria to improve interlaboratory agreement.