bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct antimicrobial susceptibility testing for acute urinary tract infections in women
Authors: Johnson J R , Tiu F S , Stamm W E
Published: January 2020

Despite its theoretical advantages, direct antimicrobial susceptibility testing (DST) of urine specimens remains controversial largely because of concerns regarding its accuracy, particularly with mixed cultures. To evaluate the performance of DST in the setting of acute urinary tract infection (UTI), we performed DST using 25 traditional and contemporary antimicrobial agents on urine specimens from 162 women with suspected acute uncomplicated UTI, and compared these results with the results of standardized disk diffusion susceptibility tests done on the same specimens. Direct tests were interpretable for 129 specimens, i.e., 80% of all specimens and 85% of the 152 specimens that met the culture criteria for UTI. Of the 2,983 individual comparisons between the direct and standard tests, 0.8% represented very major errors, 0.6% represented major errors, 3.1% represented minor errors, and 95.5% were in agreement. Errors were more common in association with older antimicrobial agents and agents with a high prevalence of antimicrobial resistance, non-Escherichia coli strains, low urine bacterial concentrations, sparse or mixed growth in the direct test, and the presence of multiple significant organisms in urine. The urine leukocyte concentration was > or = 15/mm3 in all subjects and did not differentiate between specimens that gave an interpretable direct test and those that did not. Calculation of the sensitivity of DST in identifying antimicrobial resistance supplemented conventional error rate analysis. We conclude that when used selectively and interpreted carefully, DST of urine specimens offers an efficient, rapid, and accurate method for antimicrobial susceptibility determination for acute UTI, particularly when the urine bacterial concentration is > 10(5) CFU/ml.
Detection of human immunodeficiency virus (HIV) type 1 antibodies by new automated microparticle enzyme immunoassay for HIV types 1 and 2
Authors: Matter L , Germann D
Published: January 2020

We compared an automated microparticle double-antigen sandwich enzyme immunoassay (EIA) for the IMx test system recently developed by Abbott with two established assays (the automated indirect Vidas IgG EIA and the double-antigen sandwich EIA from Murex/Wellcome) devised for the detection of human immunodeficiency virus type 1 (HIV-1) and HIV-2 antibodies. A total of 1,078 consecutive serum samples were tested prospectively with the three assays. In addition, we used retrospectively selected panels of serum samples with discrepant results in two different screening tests and with indeterminate or positive Western immunoblot (WB) results, as well as five commercially available HIV-1 seroconversion panels. The new assay showed excellent discriminatory characteristics for the separation of samples from HIV-1-positive and HIV-1-negative persons according to Centers for Disease Control and Prevention WB criteria. The sensitivities were 98.1, 92.9, and 96.1% for the new test and the two other assays, respectively, and the specificities were 99.7, 97.9, and 98.1%, respectively. With the seroconversion panels this new test was positive several days earlier than the two other assays; i.e., seroconversion was evident at the peak of p24 antigenemia and often several weeks before WB became positive by the most stringent criteria.
Evaluation of AnaeroGen system for growth of anaerobic bacteria
Authors: Miller P H , Wiggs L S , Miller J M
Published: January 2020

The Oxoid AnaeroGen system was compared with the BBL GasPak for the production of an anaerobic atmosphere and was evaluated for its ability to support the growth of 135 clinically significant anaerobic bacteria. An anaerobe chamber was used as the "gold standard" for supporting the growth of anaerobes. The AnaeroGen requires no catalyst, produces no hydrogen, requires no water, and reduces preparation time to a minimum. The water-activated BBL GasPak generates hydrogen. For 132 of the 135 strains tested, better initial growth at 48 h was noted for the jar methods than for the anaerobe chamber. At 72 h, 113 of the 135 strains showed equal growth, and at 7 days, only marginal differences in growth patterns were noted. The AnaeroGen never failed to reduce the anaerobic indicator, while the BBL GasPak occasionally failed to do so. The AnaeroGen performed at least as well as, and sometimes better than, the established methods. The AnaeroGen is a good alternative for use in anaerobic jars.
In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae
Authors: Godzik K L , O'Brien E R , Wang S K , Kuo C C
Published: January 2020

Chlamydia pneumoniae is a common respiratory pathogen. Recent studies have demonstrated the presence of C. pneumoniae in coronary and aortic atherosclerotic lesions. To study the role of C. pneumoniae in atherosclerosis, we investigated the susceptibilities of three different cells of the human vascular wall to infection with C. pneumoniae AR-39. These cell types were endothelial cells, smooth muscle cells, and macrophages derived from peripheral blood monocytes. Infection was assessed by using a direct fluorescent antibody to assess inclusion counts. Duplicate cell samples were harvested 3 days postinfection and were passed in HL cells, a susceptible human epithelial cell line, to determine if infectious organisms were produced. Endothelial cells, smooth muscle cells, and macrophages were capable of supporting C. pneumoniae growth in vitro. These results showed that three different cell types known to be important in atherogenesis are susceptible to infection with C. pneumoniae.
Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide: comparison of Bactec method with pyrazinamidase assay
Authors: Miller M A , Thibert L , Desjardins F , Siddiqi S H , Dascal A
Published: January 2020

The susceptibility of 428 clinical isolates of Mycobacterium tuberculosis to pyrazinamide was assessed by the Bactec method and the Wayne pyrazinamidase assay. The correlation between the two tests was 98.2 and 100% for susceptible and resistant strains, respectively. False resistance was seen in four (0.8%) strains with the Bactec test, and false-susceptible results occurred in two (0.5%) pyrazinamidase assays. The Bactec test is rapid and reliable, and the Bactec results correlate well with the pyrazinamidase test results, although some strains did not grow well in the test medium.
Detection of measles virus RNA in urine specimens from vaccine recipients
Authors: Rota P A , Khan A S , Durigon E , Yuran T , Villamarzo Y S , Bellini W J
Published: January 2020

Analysis of urine specimens by using reverse transcriptase-PCR was evaluated as a rapid assay to identify individuals infected with measles virus. For the study, daily urine samples were obtained from either 15-month-old children or young adults following measles immunization. Overall, measles virus RNA was detected in 10 of 12 children during the 2-week sampling period. In some cases, measles virus RNA was detected as early as 1 day or as late as 14 days after vaccination. Measles virus RNA was also detected in the urine samples from all four of the young adults between 1 and 13 days after vaccination. This assay will enable continued studies of the shedding and transmission of measles virus and, it is hoped, will provide a rapid means to identify measles infection, especially in mild or asymptomatic cases.
Production and characterization of monoclonal antibodies against Serpulina hyodysenteriae and S. innocens and their use in serotyping
Authors: Achacha M , Mittal K R
Published: January 2020

Murine monoclonal antibodies (MAbs) directed against serotypes 1, 2, 8, and 9 of Serpulina hyodysenteriae and strain B256 of Serpulina innocens were produced and characterized. A serological classification of 96 field strains of S. hyodysenteriae and 28 field strains of S. innocens isolated from pigs showing clinical signs of swine dysentery was performed by rapid dot enzyme-linked immunosorbent assay (ELISA) with the MAbs. The results indicated that the majority of the field strains of S. hyodysenteriae (69%) belonged to serotypes 8, 1, and 9, whereas only 31% of the S. innocens strains were recognized by MAb 9H7, indicating the presence of antigenic heterogeneity among S. innocens isolates. Rapid dot ELISA with type-specific MAbs was found to be specific, sensitive, and easy to perform and thus to be suitable for routine serotyping of S. hyodysenteriae and S. innocens isolates. This is the first report of MAbs being used for serotyping clinical isolates of S. hyodysenteriae and S. innocens.
Recovery of Histoplasma capsulatum from BACTEC TB media
Authors: Graham D R , Drake C , Barenfanger J E
Published: January 2020

From 1990 through 1994, we fortuitously isolated Histoplasma capsulatum from six patients with AIDS whose specimens of blood were processed by the BACTEC system using Middlebrook broth selective for acid-fast bacilli (13A medium). Growth indices became positive after an average of 17 days of incubation (range, 11 to 20 days). No acid-fast bacilli were seen, but small budding yeasts characteristic of H. capsulatum were present.
Comparison of four enzyme immunoassays for detection of human T-cell lymphotropic virus type 2 antibodies
Authors: Gallo D , Yeh E T , Moore E S , Hanson C V
Published: January 2020

Four licensed enzyme immunoassay (EIA) kits for the measurement of antibody to human T-cell lymphotropic virus (HTLV) type 1, one from Organon Teknika Corp. (OTC), one from Cambridge Biotech Corp. (CBC), and two from Abbott Laboratories (the 1993 modification [Abb 93] and the 2.0 version licensed in 1995 [Abb 95]), were evaluated for sensitivity and specificity in the detection of HTLV type 2 antibody, and the results were compared with those previously obtained with earlier kit versions. The CBC, Abb 95, Abb 93, and OTC kits had sensitivities of 99.7, 97.6, 96.8, and 96.2%, respectively, compared with sensitivities of 89.1 and 60% for the Abbott and CBC (previously DuPont) kits, respectively, licensed in 1988. Thus, the abilities of commercial kits to detect HTLV antibody have improved. The relative specificities of the CBC, Abb 95, Abb 93, and OTC kits with negative blood donor specimens that had been reactive with the 1988 CBC EIA kit were 92.9, 64.5, 78.8, and 62.6%, respectively. Compared with those of the 1988 versions, the specificity of the Abbott EIA has decreased and the specificity of the CBC kit has been significantly improved.
Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata
Authors: Pfaller M A , Houston A , Coffmann S
Published: January 2020

CHROMagar Candida is a new differential culture medium that allows selective isolation of yeasts and simultaneously identifies colonies of Candida albicans, C. tropicalis, and C. krusei. We evaluated the use of this medium with 316 yeast isolates including 247 isolated directly on CHROMagar from clinical material. Over 95% of stock and clinical isolates of C. albicans, C. tropicalis, and C. krusei were correctly identified on the basis of colony morphology and pigmentation on CHROMagar. Additionally, CHROMagar also allowed the identification of C. (Torulopsis) glabrata at a similar level of accuracy. The overall agreement between two observers in reading the CHROMagar plates was 95%. Growth of Candida sp. isolates on CHROMagar had no adverse effect on antifungal MICs or Vitek identification results. In parallel, cultures of 548 stool and rectal swab specimens set up on CHROMagar and Sabouraud glucose agar (SGA) were positive in 234 instances. CHROMagar was positive and SGA was negative for 11 specimens, and CHROMagar was negative and SGA was positive for 18 specimens. A single yeast species was isolated on both media from 162 specimens, and in 146 (90%) of these specimens the same species was detected on both CHROMagar and SGA. A total of 43 of the 234 positive cultures contained mixtures of yeast species. Twenty (47%) of these mixed cultures were detected only on CHROMagar. CHROMagar is extremely useful in making a rapid presumptive identification of common yeast species. This capability plus the ability to detect mixed cultures of Candida spp. promises to improve and streamline the work flow in the mycology and clinical microbiology laboratory.
Comparison of three commercially available enzyme-linked immunosorbent assays and biopsy-dependent diagnosis for detecting Helicobacter pylori infection
Authors: van de Wouw B A , de Boer W A , Jansz A R , Roymans R T , Staals A P
Published: January 2020

We evaluated the performance of three enzyme-linked immunosorbent assays (ELISAs) in detecting serum immunoglobulin G (IgG) and IgA antibodies to Helicobacter pylori; two were new ones from Pyloriset (Pyloriset EIA-G update and Pyloriset EIA-A update; Orion Diagnostica, Espoo, Finland), and the third was the Malakit EIA-G (Biolab, Limal, Belgium). Serum samples from 154 dyspeptic patients were collected. As a reference method, multiple biopsy specimens from different anatomical areas of the stomach were obtained by endoscopy and were analyzed by culture and/or histology and direct urease testing. Accordingly, 126 patients (82%) were found to be H. pylori positive and 28 patients (18%) were found to be H. pylori negative. To validate serology as a predictor of H. pylori infection, sensitivity, specificity, positive and negative predictive values, and accuracy of the assays were calculated against the H. pylori status as determined by the reference method. The corresponding data for the different ELISAs were 100%, 79%, 95%, 100%, and 96% for the Pyloriset ELA-G update, 81%, 89%, 97%, 52%, and 82% for the Pyloriset EIA-A update, and 87%, 86%, 96%, 60%, and 87% for the Malakit EIA-G, respectively. We conclude that the Pyloriset EIA-G update is a reliable and accurate test and that because of its 100% sensitivity, conjunctional IgA testing is not necessary. Its 100% negative predictive value makes it a very useful screening test. For purposes of excluding infection with H. pylori, the performance of the Malakit EIA-G is moderate but can be improved by conjunctional IgA testing. The Pyloriset EIA-A update can be useful as such a conjunctional test.
Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method
Authors: Holland P V , Barrera J M , Ercilla M G , Yoshida C F , Wang Y , de Olim G A , Betlach B , Kuramoto K , Okamoto H
Published: January 2020

An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype-specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes I/1a and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype II/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 4d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a.
Haemophilus influenzae adherent to contact lenses associated with production of acute ocular inflammation
Authors: Sankaridurg P R , Willcox M D , Sharma S , Gopinathan U , Janakiraman D , Hickson S , Vuppala N , Sweeney D F , Rao G N , Holden B A
Published: January 2020

Ten episodes of adverse responses to contact lens wear, including contact lens-induced acute red eye (CLARE), in which Haemophilus influenzae was isolated from contact lenses and/or from one of the external ocular sites at the time of the event, are described. All episodes occurred in patients wearing disposable hydrogel lenses on a 6-night extended-wear schedule. Two of the patients had recurrent episodes. H. influenzae was usually isolated in large numbers, and other bacteria or fungi colonizing the contact lens or the external ocular surface were usually present in low numbers. Those patients who were colonized with H. influenzae were more than 100 times as likely to have had a CLARE or infiltrative response than those subjects who were not colonized with this bacterium. H. influenzae colonization of the contact lens and eye may be subsequent to colonization of the nasopharynx because four of the seven patients presented with fever at the time of the event, with concurrent upper respiratory tract infection. Contact lens wearers should be made aware of the potential risk of CLARE associated with the wearing of contact lenses for extended periods during and subsequent to upper respiratory tract infection.
PCR ribotyping for characterizing Salmonella isolates of different serotypes
Authors: Lagatolla C , Dolzani L , Tonin E , Lavenia A , Di Michele M , Tommasini T , Monti-Bragadin C
Published: January 2020

The 16S-23S intergenic spacer region in 218 strains of Salmonella isolated from four Italian hospitals during the period from 1977 to 1994 was analyzed by PCR ribotyping. This molecular typing technique allowed for the identification of seven different and specific electrophoretic profiles for the seven serovars S: enteritidis, S. london, S. anatum, S. panama, S. heidelberg, S. agona, and S. goldcoast. Otherwise, the spacer region appears to be polymorphic in S. typhimurium S. infantis, and S. derby since we could identify eight, six, and four different ribotypes, respectively.
Comparison of pulsed-field gel electrophoresis and randomly amplified DNA polymorphism analysis for typing extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae
Authors: Gori A , Espinasse F , Deplano A , Nonhoff C , Nicolas M H , Struelens M J
Published: January 2020

The incidence and transmission patterns of extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients admitted to the intensive care unit (ICU) of a university hospital were investigated over a 3-year period. K. pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns. The discriminatory power of RAPD typing was evaluated for three primers. The incidence of isolation of ESBL-producing K. pneumoniae was 2.5 cases per 1,000 admissions to the ICU versus 0.35 cases per 1,000 admissions to other units (relative risk, 7.03; 95% confidence interval, 3.89 to 12.69). Infection developed in 53% of evaluable patients. Thirty-six percent of the cases were possibly acquired in other institutions. Isolates from ICU patients were subdivided into six capsular serotypes and into four clonal groups based on antibiotype, plasmid content, and PFGE and RAPD patterns. Two clones were associated with clusters of cross-infection, involving 5 and 12 patients, respectively. Following implementation of contact isolation precautions, the incidence of nosocomial acquisition of ESBL-producing K. pneumoniae decreased from 0.55 to 0.26 cases per 1,000 admissions (P = 0.03). PFGE and RAPD analysis showed concordant results and comparable discrimination for differentiation between groups of epidemiologically related strains of ESBL-producing K. pneumoniae. More subclonal variants were determined among epidemic clones by PFGE analysis than by RAPD analysis. Both methods are useful for typing K. pneumoniae strains in epidemiological investigations, although RAPD analysis is more efficient.
GlpQ: an antigen for serological discrimination between relapsing fever and Lyme borreliosis
Authors: Schwan T G , Schrumpf M E , Hinnebusch B J , Anderson D E , Konkel M E
Published: January 2020

Tick-borne relapsing fever is caused by numerous Borrelia species maintained in nature by Ornithodoros tick-mammal cycles. Serological confirmation is based on either an immunofluorescence assay or an enzyme-linked immunosorbent assay using whole cells or sonicated Borrelia hermsii as the antigen. However, antigenic variability of this bacterium's outer surface proteins and antigens shared with the Lyme disease spirochete (B. burgdorferi), may cause both false-negative and false-positive results when testing sera of patients suspected to have either relapsing fever or Lyme disease. To develop a specific serological test for relapsing fever, we created a genomic DNA library of B. hermsii, screened transformed Escherichia coli cells for immunoreactivity with high-titered (> or = 1:2,048) human anti-B. hermsii antiserum, and selected an immunoreactive clone (pSPR75) expressing a 39-kDa protein. DNA sequencing, subcloning, and serum adsorption experiments identified the immunoreactive protein as a homolog of GlpQ, a glycerophosphodiester phosphodiesterase identified previously in E. coli, Haemophilus influenzae, and Bacillus subtilis. Serum samples from humans and mice infected with B. hermsii or other species of relapsing fever spirochetes contained antibodies recognizing GlpQ, whereas serum samples from Lyme disease and syphilis patients were nonreactive. Serologic tests based on this antigen will identify people exposed previously to relapsing fever spirochetes and help clarify the distribution of relapsing fever and Lyme disease in situations in which the occurrence of their causative agents is uncertain.
Comparative study of three methods for genotyping hepatitis C virus strains in samples from Spanish patients
Authors: Forns X , Maluenda M D , López-Labrador F X , Ampurdanès S , Olmedo E , Costa J , Simmonds P , Sánchez-Tapias J M , Jimenez De Anta M T , Rodés J
Published: January 2020

Hepatitis C virus (HCV) genotypes may be investigated by a variety of laboratory methods that target different parts of the HCV genome and have various degrees of technical difficulty. Since the choice of a particular method is difficult, we compared the performance of (i) a type-specific PCR with type-specific primers from the core region, (ii) molecular hybridization of the PCR-amplified 5' noncoding region to type-specific probes, and (iii) identification of type-specific antibodies against epitopes of nonstructural region 4 by enzyme-linked immunosorbent assay (ELISA). One hundred fifty-one patients with biopsy-proved chronic hepatitis and HCV RNA in serum were investigated. The HCV genotype was identified in 99%, 100%, and 85% of the cases by type-specific PCR, probe hybridization, and ELISA, respectively. The type-specific PCR disclosed infection with type 1a in 3%, type 1b in 74%, and type 3a in 4% of the cases and suggested infection with two or more HCV types, including 2a/2c and 2b, in the remaining 18%. Apparently mixed infections were more prevalent in patients with past intravenous drug use (P < 0.001), but cloning and sequencing of PCR products did not confirm a mixed infection in any of the four cases investigated. Concordant results were obtained by the three procedures with virtually all of the samples in which the type-specific PCR revealed a single HCV genotype. Type-specific hybridization and ELISA usually recognized the genotype producing the strongest DNA band in samples in which type-specific PCR suggested a mixed infection. In conclusion, the three procedures evaluated in this study are reliable for investigation of HCV genotype. Type-specific PCR provides information about HCV subtypes, but a mixed infection detected with this method should be interpreted with caution.
Evaluation of Chiron HIV-1/HIV-2 recombinant immunoblot assay
Authors: Kline R L , McNairn D , Holodniy M , Mole L , Margolis D , Blattner W , Quinn T C
Published: January 2020

In a study to determine the reliability, sensitivity, and specificity of the Chiron RIBA HIV-1/HIV-2 Strip Immunoblot Assay (RIBA HIV-1/2 SIA) for confirmation of human immunodeficiency virus type 1 (HIV-1) and HIV-2 antibodies, 1,263 serum samples from various populations in the United States, Caribbean, Africa, India, and Thailand were evaluated by RIBA HIV-1/2 SIA, and the results were compared with those obtained by an HIV-1 Western blot (immunoblot) assay. All sera were tested by HIV enzyme immunoassay, RIBA HIV-1/2 SIA, and Western blotting. Samples with discrepant results were further tested by an HIV-1 and/or HIV-2 immunofluorescent-antibody assay and HIV-1 p24 antigen assay. The RIBA HIV-1/2 SIA detected all 17 HIV-1 and HIV-2 dually reactive serum samples, all 215 HIV-2-positive serum samples, and 480 of 481 HIV-1-positive serum samples for a sensitivity of 99.8%. Of 548 negative samples, 523 were RIBA HIV-1/2 SIA negative, for a specificity of 95.4%, with 22 (4%) samples interpreted as indeterminate and 3 (0.6%) interpreted as falsely positive. Western blotting detected 391 of 548 negative samples (specificity, 71.4%), with 152 (27.7%) samples interpreted as indeterminate and 5 (0.9%) interpreted as falsely positive. In conclusion, the RIBA HIV-1/2 SIA had a sensitivity comparable to that of Western blotting and could discriminate HIV-1 from HIV-2 in one blot, providing a cost advantage. Because of its high degree of specificity, the RIBA HIV-1/2 SIA further reduced the number of indeterminate results found by Western blotting, providing a more accurate means of assessing seronegative individuals.
Pseudo-outbreak of Enterococcus durans infections and colonization associated with introduction of an automated identification system software update
Authors: Singer D A , Jochimsen E M , Gielerak P , Jarvis W R
Published: January 2020

Enterococci are an important cause of hospital-acquired infections. Since 1989, there has been an increase in the number of nosocomial enterococcal infections caused by strains resistant to vancomycin in the United States. Although many enterococcal species can colonize humans, only Enterococcus faecalis, E. faecium, E. raffinosus, and E. casseliflavus have been implicated in clusters of infection. In January 1996, the Centers for Disease Control and Prevention received a report of an outbreak of vancomycin-resistant enterococci in which 31 of 84 (36.9%) isolates were identified as E. durans. Twenty-nine isolates identified as E. durans were identified to the species level after the introduction of an automated identification system software update (Vitek gram-positive identification card, version R09.1) for the identification of species of gram-positive organisms. When seven isolates initially reported as E. durans were identified to the species level by alternate methods, they were found to be E. faecium. Subsequently, isolates identified as E. durans by the automated system were reidentified by using a rapid streptococcus test, and no further enterococcal isolate has been confirmed as E. durans. Automated microbial analysis is a potential source of error that is not easily recognized. When laboratory findings are discordant with expected clinical or epidemiologic patterns, confirmatory testing by alternate methods should be performed.
Introduction of a new hepatitis agent in retrospect: genetic studies of Swedish hepatitis D virus strains
Authors: Zhang Y Y , Hansson B G
Published: January 2020

The supposedly first outbreak of hepatitis D virus (HDV) infection in Sweden occurred among intravenous drug addicts in the Malmö area in the mid-1970s. Stored sera from this outbreak were used for viral RNA extraction and analysis. By sequence comparisons, the HDV genomes from those Swedish patients fell into two separate clusters, within which the RNA sequences were closely related. These two HDV groups genetically resembled the French and US-1 isolates of genotype I, respectively, indicating that there had been at least two separate sources of HDV infection. The genetic alterations of the HDV RNA were investigated by sequence analysis of nine annually drawn serum samples from one patient and paired samples collected between 2 and more than 10 years apart from six patients with chronic HDV infection. Only mutational changes were observed, and no insertion or deletion appeared throughout the periods observed. It was found that the Swedish HDV isolates mutationally evolved at an average rate of 1.1 x 10(-3) substitutions per nucleotide per year over a long time course of chronic HDV infection, which is of the same magnitude as that of other RNA viruses.
Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R. conorii in circulating endothelial cells: a 6-year follow-up
Authors: La Scola B , Raoult D
Published: January 2020

Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever. We correlated the results of 205 R. conorii blood and skin cultures for 157 patients and the results of 48 detections of R. conorii in circulating endothelial cells (CEC) for 41 patients with relevant serological, clinical, and therapeutic data. R. conorii was cultured from 40% of patients and 29.8% of samples. R. conorii was detected in CEC in 50% of samples, representing 46.2% of patients. When these calculations were limited to the samples from untreated patients prior to their seroconversion to R. conorii, the sensitivity of culture was 59%, whereas it remained at 50% for detection in CEC. We also performed PCRs for the detection of R. conorii on eight shell vial supernatants from positive cultures and on 43 blood samples. Only nonfrozen supernatants from fresh cultures were positive. The methods described in this report are suitable for use in all laboratories. Our findings suggest that for samples to be suitable for culture they must be collected prior to the initiation of an antibiotic regimen, as early as possible in the course of the disease, and be inoculated onto shell vials with minimal delay, if R. conorii is to be successfully isolated. For patients who have been treated or who have a delayed diagnosis, detection of R. conorii in CEC remains helpful.
Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis
Authors: Shi Z Y , Liu P Y , Lau Y J , Lin Y H , Hu B S
Published: January 2020

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.
Comparison of inoculation methods for testing enterococci by using vancomycin screening agar
Authors: Jorgensen J H , McElmeel M L , Trippy C W
Published: January 2020

One hundred four recent clinical isolates of Enterococcus species were screened for vancomycin resistance by using inocula of 10(5) or 10(6) CFU dispensed by pipet and by use of a cotton swab dipped in a 0.5 McFarland standard organism suspension applied to the surface of brain heart infusion agar containing 6 micrograms of vancomycin per ml. The three inoculation methods were equivalent in the detection of nonsusceptible isolates. The use of swab inoculation was convenient and less costly than the use of micropipets.
Cloning and sequence of a region of Vibrio cholerae O139 Bengal and its use in PCR-based detection
Authors: Falklind S , Stark M , Albert M J , Uhlén M , Lundeberg J , Weintraub A
Published: January 2020

We isolated and characterized a Vibrio cholerae O139 Bengal-specific DNA region by arbitrary PCR. The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide or lipopolysaccharide biosynthesis. In order to evaluate the possibility that this region could be used for the specific detection of V. cholerae O139 Bengal, a PCR system was established. The specificity and sensitivity of the PCR were investigated by analyzing 240 strains within the family Vibrionaceae and 178 stains of other gram-negative bacteria. All V. cholerae O139 Bengal strains tested were positive, and none of the 384 control strains were amplified. The sensitivity of the assay was 10(2) CFU/ml.
Evaluation of the Digene Hybrid Capture System for detection and quantitation of human cytomegalovirus viremia in human immunodeficiency virus-infected patients
Authors: Mazzulli T , Wood S , Chua R , Walmsley S
Published: January 2020

The Digene Hybrid Capture System (DHCS) is a solution hybridization antibody capture assay for the chemiluminescent detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. This assay was compared with the CMV antigenemia assay and shell vial and tube cultures for the detection of CMV in 234 blood specimens from 72 patients with human immunodeficiency virus. Intra- and interrun precision of the DHCS assay gave coefficients of variation of 17.8 and 16.3%, respectively. The correlation coefficient for the quantitative results obtained by the DHCS assay and the antigenemia assay was 0.911 (95% confidence interval, 0.885 to 0.930). Agreement between the DHCS assay and the other three assays ranged from 83 to 86%. The DHCS assay detected 71, 87, and 84% of specimens that were positive by antigenemia, shell vial cultures, and tube culture, respectively. A total of 92% of specimens that were positive by the DHCS assay were also positive by at least one of the other assays. Evaluation of the usefulness of quantitation of CMV DNA by using the DHCS assay and its correlation with clinical disease demonstrated that, with some exceptions, patients with clinical CMV disease tended to have high levels of DNA whereas asymptomatic patients tended to have low or undetectable levels. Overall, the DHCS assay provided a rapid, quantitative, and objective measure of CMV activity in leukocytes, but results did not always correlate with clinical disease.
Performance of fungal blood cultures by using the Isolator collection system: is it cost-effective?
Authors: Morrell R M , Wasilauskas B L , Steffee C H
Published: January 2020

Enhanced recovery of fungal isolates from blood by using the Isolator system has been reported previously. We examined bacterial and fungal blood cultures during a 14-month period to determine if this enhanced recovery required a separate fungal culture and to determine the differential utility between a fungal blood culture and a routine bacterial culture. During this period, 84 of 5,196 (1.6%) fungal blood cultures and 170 of 25,702 (0.6%) bacterial blood cultures were positive for yeast or filamentous fungi. Thirty-seven positive fungal cultures, simultaneously collected, had correspondingly positive bacterial cultures. An additional 15 positive fungal cultures yielded isolates that had either been previously recovered from a bacterial culture or were recovered from a bacterial culture collected within 48 h. Of the 32 unpaired fungal cultures remaining, 5 were Candida albicans whose unique isolation was believed to be the result of specimen sampling variance rather than any enhanced recovery characteristics of fungal culture methods. Examination of patient data relating to the 27 remaining isolates (24 patients episodes) showed that only five fungal blood cultures (0.096% of all collected) had any impact on patient therapy decisions, and one of these was judged to be the cause of unnecessary therapy. Our data suggest that separate fungal cultures of blood are not cost-effective for those laboratories using the Isolator for routine blood cultures and furthermore may not be cost-effective for laboratories using automated broth systems that are comparable to the Isolator in recovery of fungi.
Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis
Authors: Yang Z , Chaves F , Barnes P F , Burman W J , Koehler J , Eisenach K D , Bates J H , Cave M D
Published: January 2020

Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments.
Prokaryotic DNA sequences in patients with chronic idiopathic prostatitis
Authors: Krieger J N , Riley D E , Roberts M C , Berger R E
Published: January 2020

Half of all men experience symptoms of prostatitis at some time in their lives, but the etiology is unknown for more than 90% of patients. Optimal clinical and culture methods were used to select 135 men with chronic prostatitis refractory to multiple previous courses of antimicrobial therapy. The subjects had no evidence of structural or functional lower genitourinary tract abnormalities of bacteriuria or bacterial prostatitis by traditional clinical tests, or of urethritis or urethral pathogens by culture. Specific PCR assays detected Mycoplasma genitalium, Chlamydia trachomatis, or Trichomonas vaginalis in 10 patients (8%). Broad-spectrum PCR tests detected tetracycline resistance-encoding genes, tetM-tetO-tetS, in 25% of patients and 16S rRNA in 77% of subjects. The tetM-tetO-tetS-positive cases constituted a subset of the 16S rRNA-positive cases. Patients with 16S rRNA were more likely to have > or = 1,000 leukocytes per mm3 in their expressed prostatic secretion than men whose prostate biopsy specimens were negative for 16S rRNA (P < 0.001). Based on direct sequencing and repetitive cloning, multiple sources of 16S rRNA were observed in individual patients. Sequences of 29 cloned PCR products revealed 16S rRNAs distinct from those of common skin and gut flora. These findings suggest that the prostate can harbor microorganisms that are not detectable by traditional approaches. These organisms may be associated with inflammation in the expressed prostatic secretions. Molecular methods hold great promise for identifying culture-resistant microorganisms in patients with chronic prostatitis.
Multicenter evaluation of a fluorometric enzyme immunocapture assay to detect toxoplasma-specific immunoglobulin M in dried blood filter paper specimens from newborns
Authors: Eaton R B , Petersen E , Seppänen H , Tuuminen T
Published: January 2020

An easy-to-perform fluorometric enzyme immunocapture assay (FEIA) was developed by Labsystems, Helsinki, Finland, to detect toxoplasma-specific immunoglobulin M (IgM) in dried blood spots. Assay materials were distributed to two sites that have programs in place designed to identify infants born with congenital toxoplasma infection: the Statens Serum Institut, Copenhagen, Denmark, and the New England Regional Newborn Screening Program, Boston, Mass. Each site tested over 700 dried blood samples from healthy newborns to define a cutoff at the 99.5 percentile (5 enzyme immunounits for Copenhagen and 4 enzyme immunounits for Boston). Each site then applied its own cutoff of interpret results for dried blood spots prepared from either adults with serology suggestive of acute infection (Copenhagen) or infants determined to be congenitally infected on the basis of serological criteria (Boston). In Copenhagen, 35 of 38 adult samples were either positive to a small degree or borderline positive for IgA. These samples thus may not represent acute infection. In Boston, of 26 congenitally infected infants, 22 were positive by FEIA. The four infant specimens not positive by FEIA were either negative or borderline positive by the standard Boston assay. These results demonstrate that the IgM FEIA is a potential alternative to other filter paper assay for toxoplasma-specific IgM currently in use for newborns.
Single-step PCR in molecular diagnosis of hepatitis C virus infection
Authors: Farma E , Boeri E , Bettini P , Repetto C M , McDermott J , Lillo F B , Varnier O E
Published: January 2020

The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection.
Differentiation of Chlamydia psittaci and C. pecorum strains by species-specific PCR
Authors: Sheehy N , Markey B , Gleeson M , Quinn P J
Published: January 2020

Sequence analyses 5' ends of the 60-kDa cysteine-rich outer membrane protein genes (Omp2) of Chlamydia psittaci and Chlamydia pecorum strains indicate that these species have approximately 70% nucleotide identity. On the basis of this sequence information, PCR primers were designed to allow the specific amplification of DNA extracted from C. psittaci S26/3 (abortion strain), P94/1 (pigeon strain), and C. pecorum W73 (fecal strain) in one reaction tube. By using nested reactions (with primers PCR-D1 and PCR-D2 followed by the specific primers and PCR-D2), 0.6, 0.2, and 8 inclusion-forming units of S26/3, P94/1 (both diluted in tissue culture-negative placental material), and W73 (diluted in culture-negative fecal material) per ml, respectively, were detected. The differentiation of C. psittaci and C. pecorum strains of ovine and bovine origins was carried out, and the results were in agreement with those obtained from AluI restriction enzyme analysis of DNA amplified from corresponding strains by PCR. This approach allows the simultaneous detection and typing of C. psittaci and C. pecorum strains and the identification of samples containing both species.
Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy
Authors: Ruhnke M , Schmidt-Westhausen A , Engelmann E , Trautmann M
Published: January 2020

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.