bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Taiwan variant of Epstein-Barr virus in throat washings from patients with head and neck tumors in Taiwan
Authors: Jeng K C , Hsu C Y , Liu M T , Chung T T , Liu S T
Published: January 2020

The prevalence of the Epstein-Barr virus (EBV) Taiwan variant was investigated in the throat washing (TW) samples from patients with head and neck tumors, persons with nonmalignant diseases, and healthy adults in Taiwan. By using the EBV (BNLF-1 gene)-specific primers and PCR, the EBV latent membrane protein gene BNLF-1 was detected in 91 (61%) of the 150 TW samples from patients with tumors, including 25 (78%) of 32 patients with nasopharyngeal carcinoma and 66 (56%) of 118 other patients with head and neck tumors. The TW samples from the 26 patients with nonmalignant tumors and 53 healthy adults were also examined. Approximately 47% of these samples were positive for the EBV gene. The PCR products of the BNLF-1 gene were then subjected to XhoI digestion. Sixty-eight of 91 PCR products (75%) showed the loss of the XhoI site, which indicated the presence of a Taiwan strain of EBV in patients with tumors. The DNA sequence of the BNLF-1 gene of the Taiwan variant revealed that the loss of the XhoI site was due to a nucleotide change from a G to a T at position 169,426 in comparison with the sequence of prototype EBV B95-8 cells. Furthermore, the Taiwan strain appeared significantly more frequently in the TWs and tissue samples from patients with nasopharyngeal carcinoma (88%; P < 0.001) and laryngeal carcinoma (80%; P < 0.02) than in those samples from healthy adults (about 40%). These data indicate that a Taiwan variant of EBV may be closely associated with head and neck tumors and suggest that this variant may be important in the pathogenesis of head and neck tumors.
Comparison of two commercially available enzyme immunoassays for detection of Clostridium difficile in stool specimens
Authors: Altaie S S , Meyer P , Dryja D
Published: January 2020

Clostridium difficile is the cause of most cases of pseudomembranous colitis, the most severe form of antibiotic-associated diarrhea. Rapid diagnosis guides both the treatment and the control of nosocomial spread of infection. Two enzyme immunoassay (EIA) kits developed for the rapid detection of C. difficile toxin A in fecal specimens, Premier (Meridian Diagnostics, Cincinnati, Ohio) and Tox-A test (TechLab, Virginia Polytechnic Institute Research Park, Blacksburg), were evaluated by using 410 fecal specimens. Seventy-six specimens were positive for C. difficile toxin B by the cytotoxin assay (prevalence rate, 19%). The Meridian EIA was positive for 71 of the 76 samples, yielding a sensitivity of 93%. The TechLab EIA detected 75 of the 76 positive samples, yielding a sensitivity of 99%. The Meridian and TechLab EIAs had specificities of 100 and 93%, respectively. These data indicate that both EIAs are suitable alternatives to the cytotoxin assay in routine diagnostic laboratories. However, confirmation of TechLab EIA-positive test results by the cytotoxin assay remains necessary.
Transmission of the human polyomavirus JC virus occurs both within the family and outside the family
Authors: Kitamura T , Kunitake T , Guo J , Tominaga T , Kawabe K , Yogo Y
Published: January 2020

JC polyomavirus (JCV) is a ubiquitous symbiote in the human population, infecting children asymptomatically and then persisting in renal tissue. We reevaluated the urinary excretion of JCV in subjects in various age groups using PCR. The detection rate for urinary JCV DNA was 9 to 17% until the age of 20 years; this rate increased dramatically to about 46% at the ages of 20 to 29 years and then increased gradually with age. Therefore, it appears that in most people excretion of JCV begins at the age of 20 to 29 years, which is earlier than suggested previously. Next, we studied the way in which JCV is spread in the human population. We selected eight Japanese families in which both parents and children excreted JCV in their urine. Their JCV subtypes were determined by PCR amplification of a JCV DNA fragment; this was followed by restriction enzyme analysis. JCV species in all JCV-positive family members were classified into either of the two subtypes, subtypes CY and MY, which are prevalent in the Japanese population. The following features of JCV subtype distribution were seen in the families: (i) both subtypes were detected in children of five of the eight families, and (ii) of 21 children who excreted JCV, 14 children excreted the same subtypes excreted by their mothers or fathers, while the remainder (7 children) excreted subtypes different from those excreted by their parents. These features suggest that JCV is transmitted both within the family and outside the family. The data also indicate that vertical transmission is not common in the spread of JCV.
Molecular typing of Cryptococcus neoformans serotype D clinical isolates
Authors: Dromer F , Varma A , Ronin O , Mathoulin S , Dupont B
Published: January 2020

Cryptococcus neoformans serotype A is responsible for the majority of cryptococcal infections in AIDS patients. In France, approximately 17% of the patients are infected with serotype D, regardless of their human immunodeficiency virus status. In a retrospective study of 273 patients, we found that serotype D was unevenly distributed in France. We wondered if this was related to the yeast's genetic background. We used karyotyping and DNA fingerprints generated by UT-4p to analyze 40 serotype D clinical isolates. We found an extensive polymorphism, with only two conserved karyotypes from drug-addicted patients living in the same area. Although highly variable, the DNA fingerprints were classified into 10 groups. Four pairs of isolates were identical; three of these pairs were from patients living in the same area, but there was no other correlation with the geographical area. The two isolates with identical karyotypes belonged to the same fingerprint group. Five of the six isolates that made up fingerprint group I were recovered from drug-addicted patients (P < 0.002; chi-square), and all five isolates found in fingerprint group III were from male homosexual patients (P < 0.02). Finally, five of the seven isolates from patients with cryptococcal pneumonia were classified as fingerprint group V (P < 0.04). These results suggest that there are possible relationships between characteristics of the isolates and body localization or even risk factors. Results of the present study warrant other studies on isolates of all serotypes and on isolates from clinical and environmental sources.
Ribosomal DNA internal transcribed spacer analysis supports synonomy of Scedosporium inflatum and Lomentospora prolificans
Authors: Lennon P A , Cooper C R , Salkin I F , Lee S B
Published: January 2020

Scedosporium inflatum is a dematiaceous opportunistic pathogen originally described by D. Malloch and I.F. Salkin (Mycotaxon 21:247-255, 1984). However, E. Gueho and G. S. De Hoog (J. Mycol. Med. 118:3-9, 1991) recently suggested reducing this mold to synonomy with Lomentospora prolificans on the basis of their similar morphological and molecular characteristics. We have investigated the ribosomal DNA internal transcribed spacers (ITS), i.e., ITS I and ITS II, of 18 isolates, including these two fungi and a closely related pathogen, Scedosporium apiospermum, and its telemorph, Pseudallescheria boydii. Identical ITS restriction fragment length polymorphisms were found in eight isolates of S. inflatum and L. prolificans. These results support Gueho and De Hoog's proposal to combine S. inflatum and L. prolificans into the binomial Scedosporium prolificans. However, the ITS I sequence of S. apiospermum and the ITS restriction fragment length polymorphisms of S. apiospermum and P. boydii were found to be significantly different from those of S. inflatum and L. prolificans. The ITS restriction pattern differences may be valuable in clinical settings for distinguishing these fungi.
Pneumococcal resistance to antimicrobial agents in the province of Québec, Canada
Authors: Jetté L P , Ringuette L , Dascal A , Lapointe J R , Turgeon P
Published: January 2020

The serogroup/serotypes (SGTs) and antimicrobial susceptibilities to 10 antimicrobial agents of 110 clinical strains of Streptococcus pneumoniae were determined. Strains intermediately resistant or highly resistant to penicillin G (80 of 110) belonged predominantly to SGTs 23 (45.0%), 19 (13.7%), 6 (10.0%), 9 (6.2%), and 14 (3.7%). The MICs of all cephalosporins, tetracycline, trimethoprim-sulfamethoxazole, and chloramphenicol increased along with the MICs of penicillin G. However, erythromycin resistance and clindamycin resistance were observed more frequently among the intermediately penicillin-resistant strains. Multiple resistance was observed for 32 strains, of which 25 were highly resistant to penicillin G and belong to SGT 23F. All strains were susceptible to vancomycin.
Rapid and sensitive method for detection of hepatitis C virus RNA by using silica particles
Authors: Cheung R C , Matsui S M , Greenberg H B
Published: January 2020

We describe a rapid, sensitive, and economic method for detection of hepatitis C virus (HCV) RNA. This method uses silica particles for purification of nucleic acid and then a modified reverse transcription-PCR that minimizes the risk of contamination and reduces the amount of reagents used. We found purification by silica particles to be at least as sensitive and in certain circumstances more sensitive than that by traditional phenol-chloroform extraction. This improved sensitivity may be due to more efficient recovery of HCV RNA by silica particles. HCV RNA appears to bind to silica particles in a saturable fashion, and the addition of extraneous nucleic acids (salmon sperm DNA or tRNA) decreases the binding in a dose-related fashion. The reverse transcription-PCR is performed by using a modified single tube method which further simplifies and reduces the cost of this assay. Finally, this method may be applied to clinical specimens such as liver tissue.
Hepatitis C virus (HCV)-specific in vitro antibody secretion by peripheral blood lymphocytes: correlation with progression of disease and HCV RNA in HCV antibody-positive patients
Authors: Ducos J , Bianchi-Mondain A M , Francois M , Boisset M , Vendrell J P , Barin F , Serre A , Larrey D
Published: January 2020

Hepatitis C virus-specific in vitro antibody production (HCV IVAP) by peripheral blood lymphocytes in 53 HCV antibody-positive patients was investigated in comparison with alanine aminotransferase (ALT) levels and HCV RNA in serum samples. All 29 HCV IVAP-positive patients were HCV RNA positive; 26 had elevated ALT levels. Among the 24 HCV IVAP-negative patients, 16 were HCV RNA negative, with 12 presenting normal ALT values. These data indicate that HCV IVAP results are highly correlated (P < 0.001) with HCV RNA results and ALT levels. Our study indicates that HCV IVAP can be used as a novel assay in the diagnosis and pathogenesis exploration of HCV infection.
Discrimination of epidemic and sporadic isolates of Acinetobacter baumannii by repetitive element PCR-mediated DNA fingerprinting
Authors: Reboli A C , Houston E D , Monteforte J S , Wood C A , Hamill R J
Published: January 2020

In 1990, there was a significant increase in the number of lower respiratory tract infections and surgical wound infections in the adult intensive care units of our tertiary care teaching hospital caused by Acinetobacter baumannii compared with the number in 1989. During the 5-month period from April through August 1990, 84 isolates of A. baumannii were recovered from 50 hospitalized patients. Biotyping, comparison of antibiograms, plasmid analysis, and DNA polymorphisms of 20 isolates from 20 different patients, determined by the use of repetitive element PCR with primers aimed at repetitive extragenic palindromic sequences and enterobacterial repetitive intergenic consensus sequences, were used to investigate this apparent outbreak. Biotyping, antibiograms, plasmid analysis, and enterobacterial repetitive intergenic consensus PCR were not useful epidemiologically. Repetitive element PCR-mediated DNA fingerprinting using repetitive extragenic palindromic primers discriminated between epidemic and sporadic strains of A. baumannii and demonstrated four discrete clusters which were unique epidemiologically.
Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy
Authors: Rådström P , Bäckman A , Qian N , Kragsbjerg P , Påhlson C , Olcén P
Published: January 2020

Primers specific to conserved and variable regions in the 16S rRNA sequence were selected from the partially sequenced 16S rRNA genes of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was divided into two DNA amplifications. The first resulted in a general bacterial amplicon, and the second resulted in a species-specific amplicon. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinical cerebrospinal fluid samples, including 125 samples from patients with bacterial meningitis, were assayed to investigate the diagnostic sensitivity and specificity for bacterial meningitis. The assay showed high sensitivity (0.94) and specificity (0.96) with the clinical samples, although some false results were obtained, the reasons for which are discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.
Bovine rotavirus V1005 a P5, not a P12, type like all viruses in a German survey
Authors: Brüssow H , Rohwedder A , Nakagomi O , Sidoti J , Eichhorn W
Published: January 2020

Bovine rotavirus (BRV) V1005, like 34 further cell culture-adapted strains in a 6-year survey in Upper Bavaria, Germany, is not a P12 but a P5 P-type rotavirus. The conclusion is based on dot blot hybridization with P1-, P5-, and P11-specific cDNAs, encompassing the VP8* region of major sequence diversity, and on PCR using P1-, P5-, and P11-specific primer pairs derived from the VP5* region of VP4 (VP5* and VP8*, respectively, are the larger and smaller tryptic cleavage products of VP4). Sequencing of the hyperdivergent region of VP4 confirmed the close relatedness of BRV V1005 to BRV UK, the P5 prototype virus.
Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates
Authors: Graves L M , Swaminathan B , Reeves M W , Hunter S B , Weaver R E , Plikaytis B D , Schuchat A
Published: January 2020

Ribotyping was compared with multilocus enzyme electrophoresis (MEE) for subtyping 305 Listeria monocytogenes isolates from clinical and nonclinical sources. For ribotyping, EcoRI-restricted genomic DNA fragments of L. monocytogenes strains were separated by agarose gel electrophoresis, and Southern blots were probed with a cloned Escherichia coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. The L. monocytogenes isolates were divided into 28 distinct ribotypes, while MEE analysis divided the same isolates into 78 electrophoretic types (ETs). On the basis of their ribotype profiles, the strains were divided into two subgroups. The ribotype alpha (RT alpha) subgroup contained serotypes 1/2a, 1/2c, and 3a, and the ribotype beta (RT beta) subgroup contained serotypes 1/2b, 3b, 4b, and 4ab. This division is in complete agreement with MEE analysis, which divides the species into two subgroups (ET groups A and B), with the same serotype distribution in each subgroup. Overall, MEE was more discriminating than ribotyping. However, in several instances ribotyping discriminated between isolates within the same ET. Ribotyping was more discriminating for serotypes 1/2a, 1/2c, and 3a (Simpson's Index for Diversity [DI] = 0.81) than for serotypes 1/2b and 4b (DI = 0.76). A substantial proportion (69%) of serotype 1/2b and 4b strains clustered in five ETs and five ribotypes. These data suggest that ribotyping and MEE do not provide adequate discrimination between strains of serotypes 1/2b and 4b. Methods such as pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis should be explored for further discrimination of strains of these serotypes.
Molecular fingerprinting of Legionella species by repetitive element PCR
Authors: Georghiou P R , Doggett A M , Kielhofner M A , Stout J E , Watson D A , Lupski J R , Hamill R J
Published: January 2020

Repetitive element PCR (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species. Substantial variation was seen among the rep-PCR fingerprints of different Legionella species and serogroups. More limited, but distinct, polymorphisms of the rep-PCR fingerprint were evident among epidemiologically unrelated isolates of L. pneumophila serogroup 1. Previously characterized Legionella isolates from nosocomial outbreaks were correctly clustered by this method. These results suggest the presence of repetitive extragenic palindromic-like elements within the genomes of members of the family Legionellaceae that can be used to discriminate between strains within a serogroup of L. pneumophila and between different Legionella species. rep-PCR appears to be a useful technique for the molecular fingerprinting of Legionella species.
Antigenic, morphologic, and molecular characterization of new Ehrlichia risticii isolates
Authors: Chaichanasiriwithaya W , Rikihisa Y , Yamamoto S , Reed S , Crawford T B , Perryman L E , Palmer G H
Published: January 2020

Ehrlichia risticii causes an acute infectious disease in horses called Potomac horse fever. To investigate the biological diversity of E. risticii organisms, nine E. risticii isolates derived from the peripheral blood monocytes of clinically sick horses in Ohio and Kentucky during the summers of 1991 and 1993 were compared with Illinois and Virginia isolates originally obtained from horses in Maryland in 1984. Seven of the nine isolates (081, 606, 380, 679, As, Co, and Ov) formed large morulae (tightly packed inclusions of ehrlichial organisms). The remaining isolates, including 1984 isolates, were individually dispersed or formed small morulae in the cytoplasm of P388D1 cells. In Western blot (immunoblot) analysis with four equine and one rabbit polyclonal anti-E. risticii sera, these recent E. risticii isolates showed patterns of antigenic proteins distinct from those of the 1984 isolates and could be divided into three groups: (i) 081; (ii) 606, 022, 067, 380, and 679; and (iii) As, Co, and Ov. By indirect fluorescent antibody labeling with two panels of murine anti-E. risticii (Illinois and Maryland isolates) monoclonal antibodies, isolate 081 was not labeled with any of 20 monoclonal antibodies tested. The remaining isolates were not labeled with several monoclonal antibodies. The digestion pattern with one of the restriction enzymes, AvaII, of the PCR-amplified partial 16S rRNA gene of E. risticii from all Kentucky isolates (As, Co, and Ov) was different from that of Illinois, Virginia, and six Ohio isolates. These results indicate the presence of distinct variants of E. risticii which vary significantly in morphology, antigenic composition, and the base sequence of the 16S rRNA gene.
Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms
Authors: Zimmermann C R , Snedker C J , Pappagianis D
Published: January 2020

The marked increase in the number of cases of coccidioidomycosis in California in 1992 led to a study of isolates from various patients and environmental sources by restriction fragment length polymorphism (RFLP) analysis. Of 15 different isolates, most of the isolates (13 of 15) from California and 1 from Venezuela yielded one main RFLP pattern with evidence of two subgroups. The other two isolates (both from patients in the San Joaquin Valley of California) yielded a different RFLP pattern.
Optimization of quantitative culture assay for human immunodeficiency virus from plasma. Plasma Viremia Group Laboratories of the AIDS Clinical Trials Group (National Institute of Allergy and Infectious Diseases)
Authors: Lathey J L , Fiscus S A , Rasheed S , Kappes J C , Griffith B P , Elbeik T , Spector S A , Reichelderfer P S
Published: January 2020

Experimental conditions essential to a simple, reproducible, quantitative human immunodeficiency virus plasma assay were determined. Five parameters were evaluated: length of culture, reproducibility, assay dilution schema, washing procedures, and anticoagulant usage. The recommended quantitative plasma assay utilizes undiluted citrated plasma cultured with peripheral blood mononuclear cells for 14 days with fivefold dilutions and a medium change on day 1 with no washing.
Severe osteomyelitis due to the zygomycete Apophysomyces elegans
Authors: Meis J F , Kullberg B J , Pruszczynski M , Veth R P
Published: January 2020

We describe a previously healthy 69-year-old man presenting with osteomyelitis of the humerus due to the zygomycete Apophysomyces elegans. The infection was acquired in Aruba, The Netherlands Antilles. The skin provided the most likely portal of entry, although there was no history of a traumatic inoculation. The patient had no history of diabetes, and no underlying immune defects were found. Despite treatment with 7.9 g of amphotericin B, an interthoracoscapular amputation proved necessary to curtail the rapid spread of the fungus in this immunocompetent host.
Sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis C virus in The Netherlands
Authors: Kleter G E , van Doorn L J , Brouwer J T , Schalm S W , Heijtink R A , Quint W G
Published: January 2020

The RNAs of hepatitis C virus (HCV) isolates from 62 patients with chronic HCV infection were analyzed by direct sequencing of the 5' untranslated region. Two important sequence motifs were recognized: one between positions -170 and -155 and the other between positions -132 and -117. These motifs are partly complementary. All three previously published genotypes were observed; 34 (55%) isolates were classified as type 1 (including prototype [from the United States] and HCV-BK [from Japan] sequences), 11 (18%) were classified as type 2 (including HC-J6 and HC-J8), and 12 (19%) were classified as type 3 (including EB1); one patient was infected with genotypes 1 and 2. Four (6%) isolates showed aberrant sequences and were therefore provisionally classified as genotype 4. These results indicate the significance of sequence variation among the 5' untranslated regions of different HCV genotypes and indicate that this region could possibly be used for consistent genotyping of HCV isolates.
Diagnosis of human immunodeficiency virus (HIV) infection: multicenter evaluation of a newly developed anti-HIV 1 and 2 enzyme immunoassay
Authors: Hess G , Avillez F , Lourenco M H , D'Agostino F , Cambie G , Piot P , Vercauteren G , Michl U , Melchior W , Bayer H
Published: January 2020

A new anti-human immunodeficiency virus type 1 and 2 (anti-HIV 1 and 2) test is described. It uses recombinant p24 and peptides covering gp32, gp41, and gp120 to identify HIV-1 and HIV-2 infections. This test has been shown to be specific (99.5%) and sensitive (99.8%). In this respect, the assay was equal or superior to anti-HIV 1 and 2 tests run as references. The test was able to discriminate sera from patients with HIV infections from those from uninfected individuals with excellence; it also exerted high intra- and interassay precisions. The "modular" concept of the test allows the use of single components (gp32 or gp41) to separate between HIV-2 and HIV-1 infections, respectively.
Genotypic variation and slime production among blood and catheter isolates of Candida parapsilosis
Authors: Branchini M L , Pfaller M A , Rhine-Chalberg J , Frempong T , Isenberg H D
Published: January 2020

Candida parapsilosis is an important nosocomial pathogen that can proliferate in high concentrations of glucose and form biofilms on prosthetic materials. We investigated the genotypic diversity and slime production among 31 isolates of C. parapsilosis from individual patients with bloodstream or catheter infections. DNA subtyping was performed by using electrophoretic karyotyping plus restriction endonuclease analysis with BssHII followed by pulsed-field gel electrophoresis. Slime production was evaluated by growing organisms in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer. Overall there were 14 DNA subtypes among the 31 isolates. Eighty percent of the isolates produced slime; 67% of the isolates were moderately to strongly positive, 13% were weakly positive, and 20% were not slime producers. The ability of isolates of a given DNA type to produce slime under these conditions was variable. The results of these studies indicate moderate genotypic variation among clinical isolates of C. parapsilosis. The propensity of these isolates to form slime in glucose-containing solutions suggests that this phenotypic characteristic may contribute to the ability of C. parapsilosis to adhere to plastic catheters and cause infections.
Rapid detection of parainfluenza virus type 3 RNA in respiratory specimens: use of reverse transcription-PCR-enzyme immunoassay
Authors: Karron R A , Froehlich J L , Bobo L , Belshe R B , Yolken R H
Published: January 2020

Parainfluenza virus type 3 (PIV-3), an important lower respiratory tract pathogen in young children and immunocompromised individuals, may be underdiagnosed because of the insensitivity of available culturing systems and delay in identification of virus in cell culture. We developed a reverse transcription-PCR-enzyme immunoassay (RT-PCR-EIA) for PIV-3, using primers specific for a highly conserved region of the hemagglutinin-neuraminidase gene. Testing of nasal washes spiked with PIV-3 or other respiratory viruses showed that this assay detected seven strains of PIV-3 but not other respiratory viruses. Of 103 respiratory tract samples obtained from children experimentally infected with a liver PIV-3 vaccine or naturally infected with wild-type PIV-3, 51 were positive by culture and 48 were positive by RT-PCR-EIA. Eleven of the culture-positive samples were negative by RT-PCR-EIA; however, none of these grew virus upon reinoculation into cell culture, indicating that virus was lost or was present at a very low titer. Eight of the culture-negative samples were positive by RT-PCR-EIA: two were obtained from a subject who was culture negative but had a serologic response to PIV-3, four were obtained 7 to 9 days after the first positive culture, and two were obtained 1 day prior to the first positive culture. Thus, this RT-PCR-EIA for PIV-3 is sensitive and specific and can detect viral RNA in samples from which virus cannot be cultivated. This assay could be used for diagnosis late in the course of PIV-3 infection and for accurate detection of disease outbreaks.
Isolation of hepatitis C virus RNA from serum for reverse transcription-PCR
Authors: Nolte F S , Thurmond C , Mitchell P S
Published: January 2020

Standard multistep extraction and isolation of RNA for hepatitis C virus (HCV) reverse transcription (RT)-PCR are impractical for routine use in clinical laboratories. We compared three simple commercially available methods for RNA isolation (RNAzol B, TRISOLV, and ULTRASPEC; Biotecx Laboratories, Houston, Tex.) and a total nucleic acid isolation method (IsoQuick; MicroProbe Corp., Garden Grove, Calif.) for the recovery of HCV RNA from sera obtained from 12 viremic patients for RT-PCR. RNAzol B, TRISOLV, ULTRASPEC, and IsoQuick extraction methods detected 87.5, 79.2, 33.3, and 58.3% of the paired positive samples, respectively. The method used for isolation of RNA is an important concern when optimizing HCV RT-PCR.
Identification of Streptococcus suis isolated from swine: proposal for biochemical parameters
Authors: Tarradas C , Arenas A , Maldonado A , Luque I , Miranda A , Perea A
Published: January 2020

A study was made of the biochemical profiles of 59 strains serotyped as Streptococcus suis, isolated from diseased and clinically healthy pigs. The following parameters are proposed for the identification of the species: Voges-Proskauer negativity, hydrolysis of esculin positivity, trehalose positivity, negativity for growth in 6.5% NaCl, and absence of beta-hemolysis on sheep blood agar. S. suis serotype 2 is negative for hippurate, pyrrolidonylarylamidase, and mannose.
Antimicrobial susceptibility of Staphylococcus hyicus isolated from exudative epidermitis in pigs
Authors: Wegener H C , Watts J L , Salmon S A , Yancey R J
Published: January 2020

Exudative epidermitis or greasy pig syndrome is caused by the coagulase-variable staphylococcal species Staphylococcus hyicus. Treatment of this disease is problematic because of the limited number of antimicrobial agents available for this purpose. Thirteen antimicrobial agents were evaluated for their activities against 100 S. hyicus strains isolated from pigs with exudative epidermitis. Novobiocin was the most active compound tested, with an MIC for 90% of the strains tested (MIC90) of < or = 0.06 microgram/ml. Enrofloxacin, ampicillin, and ceftiofur were the next most active compounds, with MIC90s of 0.25, 0.5, and 1.0 microgram/ml, respectively. However, 41.4% of the 99 strains tested were positive for beta-lactamase production. The MIC90s of erythromycin, tetracycline, and streptomycin were > 32.0 micrograms/ml. Initial testing with sulfadiazine-trimethoprim yielded an MIC90 of > 64.0 micrograms/ml, but subsequent testing with thymidine phosphorylase-supplemented medium yielded an MIC90 of 0.06 microgram/ml. Both lincomycin and spectinomycin were relatively inactive against the S. hyicus strains tested, with MIC90s of > 64.0 and > 128.0 micrograms/ml, respectively. However, the combination of the two compounds at ratios of 1:2 (lincomycin to spectinomycin) and 1:8 were more active, with MIC90s of 16.0 and 4.0 micrograms/ml, respectively. These results indicate that novobiocin and sulfadiazine-trimethoprim were the most active compounds tested against the S. hyicus strains isolated from pigs with exudative epidermitis. Furthermore, the combination of lincomycin and spectinomycin was more active than the individual compounds against the strains tested.
Factors influencing detection of quantitative cytomegalovirus antigenemia
Authors: Boeckh M , Woogerd P M , Stevens-Ayers T , Ray C G , Bowden R A
Published: January 2020

Of 20 blood specimens testing positive for cytomegalovirus antigen after immediate processing, 19 (95%) remained positive when kept at room temperature for 24 h before processing. Quantitative antigenemia decreased by an average of 44% after storage. Compared with acetone fixation, formaldehyde fixation showed improved readability, fewer artifacts, and a higher degree of sensitivity.
Accuracy and cost of antibiotic susceptibility testing of mixed morphotypes of Pseudomonas aeruginosa
Authors: Morlin G L , Hedges D L , Smith A L , Burns J L
Published: January 2020

Chronic Pseudomonas aeruginosa colonization of the lower respiratory tract of patients with cystic fibrosis frequently results in pulmonary exacerbations requiring treatment with antimicrobial agents. Multiple morphotypes with different antibiotic susceptibilities are often isolated from a single sputum sample. Determination of MICs of antibiotics for each sputum morphotype is used to guide therapy but is time-consuming and expensive. We explored an alternative assay for determining MICs for all P. aeruginosa morphotypes cultured from a homogenized sputum sample. We sought correlations of those MICs with the MIC for the most resistant morphotypes tested separately. The MICs determined for a mixture of morphotypes correctly predicted the highest MICs (+/- one dilution) determined for isolated morphotypes 73.5% of the time. The MIC for the mixed morphotypes correctly predicted susceptibility in 90.4% of samples. In contrast, determination of the MIC for the mixture of morphotypes correctly predicted resistance in only 57.0%. For sputa containing susceptible isolates, testing the mixed culture may provide adequate susceptibility data with significant laboratory time and cost savings. However, for sputa with resistant strains, the traditional method of testing isolated morphotypes should still be used.
Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas
Authors: Kapur V , Li L L , Iordanescu S , Hamrick M R , Wanger A , Kreiswirth B N , Musser J M
Published: January 2020

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.
Importance of the fiberoptic endoscope cleaning procedure for detection of Helicobacter pylori in gastric biopsy specimens by PCR
Authors: Roosendaal R , Kuipers E J , van den Brule A J , Peña A S , Uyterlinde A M , Walboomers J M , Meuwissen S G , de Graaff J
Published: January 2020

A 16S ribosomal DNA-based PCR appeared to be a sensitive test for the detection of infection by Helicobacter pylori in 31 patients when compared with culturing and histological and serological techniques. For five patients, PCR was the only test with a positive result. H. pylori DNA was also found in gastrointestinal equipment even after standard intensive combined manual and machine cleaning. We therefore conclude that a reliable validation of PCR for the detection of H. pylori in gastric biopsy specimens is possible only when the cleaning and disinfection method used has been proven to remove all H. pylori DNA from gastrointestinal equipment. An adequate cleaning and disinfection method for the removal of H. pylori DNA from fiberoptic endoscopes is described.
Diagnostic utility of PCR-enzyme immunoassay, culture, and serology for detection of Chlamydia pneumoniae in symptomatic and asymptomatic patients
Authors: Gaydos C A , Roblin P M , Hammerschlag M R , Hyman C L , Eiden J J , Schachter J , Quinn T C
Published: January 2020

To assess the utility of PCR-enzyme immunoassay (EIA) for diagnosis of acute infection with Chlamydia pneumoniae, we compared tissue culture, PCR-EIA, direct fluorescent-antibody (DFA) stain, and serology in studies with 56 patients with respiratory symptoms and 80 asymptomatic persons. Thirty-five patients were positive by either culture or PCR-EIA, and 101 were negative by both assays. Thirty specimens from symptomatic patients and one from an asymptomatic patient were culture positive; 23 of these were also PCR-EIA positive. Of the eight culture-positive, PCR-EIA-negative specimens, five were DFA negative and three were DFA positive. Four additional specimens were culture negative and PCR-EIA positive; of these, three were DFA positive and one was DFA negative. When we used culture- and/or DFA-positive results as a reference or "gold standard," the sensitivity and specificity of PCR were 76.5 and 99.0%, respectively. When we used PCR- and/or DFA-positive results as the reference, the sensitivity of culture was 87.5%. On the basis of single acute serum specimens, only 8 of these 35 patients had diagnostic antibody titers. Of the asymptomatic patients, 75% had immunoglobulin G or immunoglobulin M antibody to C. pneumoniae; 15 (18.8%) of these had antibody levels considered to be diagnostic of acute infection. This multicenter study indicates that culture and/or PCR-EIA is more reliable for prompt diagnosis of C. pneumoniae infection than single-point serology alone.
Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA
Authors: Savio M L , Rossi C , Fusi P , Tagliabue S , Pacciarini M L
Published: January 2020

Primers for PCR were selected from a sequenced fragment of clone pL590, which contains a repetitive element present in the genome of Leptospira interrogans serovar hardjo type hardjoprajitno (M. L. Pacciarini, M. L. Savio, S. Tagliabue, and C. Rossi, J. Clin. Microbiol. 30:1243-1249, 1992). A specific DNA fragment was amplified from the genomic DNAs of serovar hardjo type hardjoprajitno and nine serovars also belonging to L. interrogans as a consequence of the spread of the same or a closely related repetitive element within this species (Pacciarini et al., J. Clin. Microbiol. 30:1243-1249, 1992). In addition, specific amplification was obtained from two Leptospira borgpetersenii serovars (tarassovi and hardjo type hardjobovis). Negative PCR results were observed with all of the other Leptospira serovars tested, including nonpathogenic ones (serovars patoc and andamana), another spirochete (Borrelia burgdorferi), bacteria commonly found in biological samples, and swine and bovine cell lines. Direct PCR on biological samples such as kidney samples demonstrated that preliminary isolation and culture of Leptospira cells are not required for efficient detection. Furthermore, digestion of the amplified DNA with the enzymes HinfI and DdeI yielded specific polymorphic patterns, allowing discrimination among the majority of the serovars. These methods were applied to 25 field isolates of serovar pomona, leading to the conclusion that they were suitable for the simple and rapid detection of L. interrogans and for serovar identification.
Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR
Authors: Anderson B , Sims K , Regnery R , Robinson L , Schmidt M J , Goral S , Hager C , Edwards K
Published: January 2020

A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R. quintana. Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species. DNAs from 12 different isolates of R. henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R. henselae-specific probe. DNAs from four different isolates of R. quintana were amplified and produced a PCR product of the same size that hybridized only with the R. quintana-specific probe. DNAs from isolates of R. elizabethae, R. vinsonii, Bartonella bacilliformis, and Afipia felis failed to amplify the 414-bp fragment in the PCR assay. This two-step assay was applied to DNAs extracted from 16 fresh (unfixed) lymph node biopsy specimens and nine aspirates from patients with clinical cat scratch disease (CSD) to assay for the presence of R. henselae or R. quintana DNA in these samples. Twenty-one (84%) of 25 lymph node samples from CSD patients were positive for R. henselae, while none were positive for R. quintana. The characteristic 414-bp fragment was not amplified from eight lymph node tissue samples from non-CSD cases. These results provide evidence that R. henselae, and not R. quintana, plays the central role in the etiology of CSD.
Investigation of the sequence of colonization and candidemia in nonneutropenic patients
Authors: Voss A , Hollis R J , Pfaller M A , Wenzel R P , Doebbeling B N
Published: January 2020

Among neutropenic patients with hematologic malignancies, candidemia has been shown to arise typically from autoinfection after colonization. In patients without neutropenia, we examined the similarities of strains colonizing or infecting various body sites and those subsequently causing Candida bloodstream infections. Strain similarity was examined by karyotyping and restriction endonuclease analysis of genomic DNA (REAG) by using two restriction enzymes (SfiI and BssHII). The banding patterns of 42 isolates from 19 patients were independently evaluated in a blinded fashion by three observers. The interobserver reliability measured with a generalized kappa statistic was 0.59 for karyotyping, 0.84 for REAG with SfiI, and 0.88 for REAG with BssHII (P < 0.001 for each). REAG classified the initial colonizing or infecting isolate and subsequent blood isolates as identical in 16 patients (84%). The mean duration of colonization or infection prior to a positive blood culture was 5 and 23 days in patients infected with related and unrelated isolates, respectively (P = 0.14; 95% confidence interval = -14.5 to 50.5). Karyotyping results matched the REAG results for isolates from 14 of the 19 patients (74%). In patients infected with identical isolates, the initial isolate was most frequently recovered from the urine (n = 5) or vascular catheter tips (n = 4). In the five subjects with organisms showing disparate results between the methods, karyotyping revealed different banding patterns, whereas REAG suggested that the isolates were identical. Candida colonization or infection with an identical strain frequently precedes bloodstream infection in nonneutropenic patients. Future studies should evaluate whether patients at high risk for candidemia and who have vascular catheter or urine samples that are positive for a Candida on culture should be treated empirically.
Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice
Authors: Fox J G , Dewhirst F E , Tully J G , Paster B J , Yan L , Taylor N S , Collins M J , Gorelick P L , Ward J M
Published: January 2020

A bacterium with a spiral shape and bipolar, single, sheathed flagella was isolated from the livers of mice with active, chronic hepatitis. The bacteria also colonized the cecal and colonic mucosae of mice. The bacterium grew at 37 degrees C under microaerophilic and anaerobic conditions, rapidly hydrolyzed urea, was catalase and oxidase positive, reduced nitrate to nitrite, and was resistant to cephalothin metronidazole. On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel helicobacter, Helicobacter hepaticus. This new helicobacter, like two other murine Helicobacter species, H. muridarum and "H. rappini," is an efficient colonizer of the gastrointestinal tract, but in addition, it has the pathogenic potential to elicit persistent hepatitis in mice.
Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of gram-negative bacteria
Authors: Baker C N , Banerjee S N , Tenover F C
Published: January 2020

The Alamar (Alamar Biosciences, Inc., Sacramento, Calif.) colorimetric antimicrobial susceptibility testing method is a new approach to the determination of broth microdilution MICs. The method uses a color indicator to detect growth of microorganisms within the wells of a microdilution tray. The color changes can be read visually or with a fluorometer. The system contains growth and sterility control wells and 20 antimicrobial agents per MIC tray with eight twofold dilutions for each antimicrobial agent. We tested 186 multiresistant, gram-negative bacterial isolates against 33 antimicrobial agents and compared the results to those obtained by agar dilution. Categorical agreement for all agents was 90.9% and ranged from 78.2% for ampicillin-sulbactam to 98.1% for amikacin. Percent agreement for MIC results (within +/- 1 log2 dilution) was 91.0% for all agents and ranged from 69.1% for gentamicin to 97.9% for ciprofloxacin. Most of the disagreements were with the penicillins and cephalosporins for beta-lactamase-producing strains. The Alamar MIC system is very easy to read visually and appears to be a satisfactory addition to currently used MIC determination methods.
Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified polymorphic DNA patterns
Authors: Czajka J , Batt C A
Published: January 2020

Food and clinical isolates of Listeria monocytogenes recovered from four different outbreaks of listeriosis were analyzed by their PCR-based randomly amplified polymorphic DNA (RAPD) patterns to verify their causal relationships. The generation of DNA fingerprints by PCR-based RAPD analysis is a fast and sensitive method for the epidemiological tracking and identification of bacteria implicated in food poisoning outbreaks. The L. monocytogenes strains used in the study were obtained from the following four outbreaks: California, 1985, Mexican-style cheese; Canadian Maritime Provinces, 1981, coleslaw; Canada, 1989, brie cheese; and Canada, 1989, alfalfa tablets. RAPD profiles were generated by using random 10-mer primers for at least one food and one clinical isolate recovered from each outbreak. Identical profiles for 20 different primers were observed for each pair of food and clinical isolates from two of the four outbreaks. Isolates from the outbreak involving alfalfa tablets exhibited identical patterns for 19 primers; however, primer OPA-1 produced one additional 1.8-kb fragment, designated OPA-1-1.8, that was found in the food isolate but not in the corresponding clinical isolate. Hybridization analysis revealed that the absence of the OPA-1-1.8 polymorphic fragment in the clinical isolate was due to a deletion of at least 1.8 kb. Loss of the OPA-1-1.8 polymorphic fragment could not be induced by infective passage of the L. monocytogenes isolate from the alfalfa tablet through a mouse or by growth of this isolate under selective conditions. This suggests that the isolate recovered from the food was not identical to the isolate recovered from the patient. The ability to produce unique RAPD patterns allows for the discrimination between isolates even if they are of the same serotype and multilocus enzyme electrophoretic type.
Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli
Authors: Grewal H M , Helander A , Svennerholm A M , Bhan M K , Gaastra W , Sommerfelt H
Published: January 2020

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.