bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of DNA probes for fingerprinting Aspergillus fumigatus
Authors: Girardin H , Latgé J P , Srikantha T , Morrow B , Soll D R
Published: January 2020

Several different DNA fragments containing nonribosomal repetitive sequences have been isolated from the genome of Aspergillus fumigatus and tested as potential DNA fingerprinting probes. Eight of these clones generate 19 or more bands when hybridized to EcoRI-digested DNA of a reference strain in Southern blots, and they fall into four families. Individual clones from two families were tested and were found to generate complex Southern blot hybridization patterns which are stable within a single strain over many generations, which vary among unrelated strains, and which are amenable to computer-assisted analyses involving large numbers of strains in epidemiological studies. Clones from three of the families clustered a majority of test strains in a similar fashion in individual dendrograms based on similarity coefficients computed from band positions in Southern blot hybridization patterns. These clones therefore fulfill the major requisites for effective DNA fingerprinting probes.
Relationships among Pseudomonas pseudomallei isolates from patients with recurrent melioidosis
Authors: Desmarchelier P M , Dance D A , Chaowagul W , Suputtamongkol Y , White N J , Pitt T L
Published: January 2020

Patients with melioidosis may present with recurrent infections after clinical resolution of their primary illness. Because there has been no satisfactory typing scheme for Pseudomonas pseudomallei, recrudescence could not be distinguished from reinfection. We determined the strain identity of primary and relapse isolates of P. pseudomallei from 25 patients with culture-proven melioidosis to answer whether secondary infections were due to the initial infecting strain or to the acquisition of a new strain. Fifty-four isolates were compared by the patterns of BamHI restriction digests produced after hybridization with a cDNA copy of Escherichia coli rRNA. Twenty-three patients had primary and relapse isolates with identical or highly similar ribotype patterns. The patterns of isolates from two patients were different; the primary and relapse isolates differed by a single fragment for one, and the other had identical primary and first-relapse isolates while the second-relapse isolate was markedly different. The results indicated that recurrent infection probably resulted from endogenous relapse in most of the melioidosis patients studied, although reinfection from an exogenous source was also possible in two cases.
Clonal spread of vancomycin-resistant Enterococcus faecium between patients in three hospitals in two states
Authors: Chow J W , Kuritza A , Shlaes D M , Green M , Sahm D F , Zervos M J
Published: January 2020

The DNAs of 3* vancomycin-resistant Enterococcus faecium isolates from five hospitals in three states were analyzed by contour-clamped homogeneous electric field electrophoresis and plasmid analysis. There were 22 strain types. One strain type was common to patients in three hospitals in two states. These results suggest the apparent intra- and interhospital spread of vancomycin-resistant E. faecium.
Resolutive Candida utilis fungemia in a nonneutropenic patient
Authors: Bougnoux M E , Gueho E , Potocka A C
Published: January 2020

We report here the second case of Candida utilis infection in humans. The patient was apparently immunocompetent, had no central catheter, and survived an 8-day fungemia. Genomic analysis confirmed the conspecificity of medical and industrial strains of C. utilis and that of the anamorphic yeast C. utilis with the teleomorphic yeast Pichia jadinii.
Increased detection of human immunodeficiency virus antigenemia after dissociation of immune complexes at low pH
Authors: Pokriefka R A , Manzor O , Markowitz N P , Saravolatz L D , Kvale P , Donovan R M
Published: January 2020

This study compared the number of patients with detectable human immunodeficiency virus (HIV) antigenemia after immune complex (IC) dissociation by established methods using either 0.5 NCl or 1.5 M glycine buffer. Without IC dissociation, HIV antigen was detected in 43% of patients. After dissociation, the HCl method detected only an additional 7% of patients (P = 0.09), while the glycine method detected an additional 34% (P < 0.001). However, care must be taken in setting the threshold of the standards, and confirmatory neutralization assays should be performed to ensure specificity of HIV antigen enzyme immunoassay after IC dissociation.
Coryneform bacteria isolated from middle ear fluid
Authors: Simonet M , De Briel D , Boucot I , Minck R , Veron M
Published: January 2020

Nineteen strains of facultatively anaerobic gram-positive rods isolated in pure culture from middle ear fluids were identified. All effusions were collected by tympanocentesis in children with acute otitis media. Identification of microorganisms to the genus level was done by studying the cell wall composition. Sixteen strains contain meso-diaminopimelic acid and arabinogalactan polymer but lack mycolic acids; therefore, these strains do not belong to a previously described taxon. Because of similarities with Corynebacterium afermentans (Centers for Disease Control group ANF-1), we temporarily classified these mycolateless strains ANF-1 like. Isolation of these microorganisms in pure culture from middle ear fluids collected by tympanocentesis is a strong argument for their involvement in acute otitis media.
Unrelatedness of groups of yeasts within the Candida haemulonii complex
Authors: Lehmann P F , Wu L C , Pruitt W R , Meyer S A , Ahearn D G
Published: January 2020

Isoenzyme and protein profiles of clinical isolates initially identified as Candida haemulonii demonstrated the presence of two distinct groups. DNA relatedness studies with representative cultures confirmed the presence of two species. Physiological features that can be used to separate two groups within C. haemulonii are reported.
Polymerase chain reaction-based restriction fragment length polymorphism analysis of a fragment of the ribosomal operon from Rochalimaea species for subtyping
Authors: Matar G M , Swaminathan B , Hunter S B , Slater L N , Welch D F
Published: January 2020

Restriction endonuclease analysis of a polymerase chain reaction-amplified DNA fragment which included the spacer region between the genes coding for 16S and 23S rRNAs and a portion of the gene coding for 23S rRNA (spacer + 23S) was done on 10 previously characterized clinical isolates of Rochalimaea henselae, one clinical isolate of Rochalimaea quintana, and the type strains of R. henselae, R. quintana, Rochalimaea vinsonii, and Bartonella bacilliformis. Brucella abortus DNA was not amplified by the primer set used. The clinical isolates of Rochalimaea were obtained from blood or tissue from patients with and without preexisting disease. The amplicon from each strain was digested with five endonucleases (AluI, HaeIII, TaqI, HinfI, and MseI). AluI and HaeIII were useful in species differentiation and subtyping of R. henselae. R. henselae isolates showed six different restriction patterns with AluI and four patterns with HaeIII. TaqI, HinfI, and MseI were useful only in species differentiation. These observations indicate that PCR amplification of the spacer + 23S region of the ribosomal DNA of Rochalimaea spp., along with restriction endonuclease analysis, allows differentiation of Rochalimaea spp. from closely related genera, differentiation among the species within Rochalimaea, and differentiation of strains within R. henselae. The subtyping potential of this method may be useful for further clinical and epidemiologic studies of the spectrum of diseases caused by R. henselae.
Distribution of VP4 gene alleles in human rotaviruses by using probes to the hyperdivergent region of the VP4 gene
Authors: Steele A D , Garcia D , Sears J , Gerna G , Nakagomi O , Flores J
Published: January 2020

The rotavirus VP4 protein elicits the production of neutralizing antibodies and is known to play a role in inducing resistance to disease. At least five human rotavirus VP4 gene alleles have been described on the basis of antigenic polymorphism and/or nucleotide sequence differences. In the present study, we developed cDNA probes directed at the hyperdivergent region of the VP4 gene of the five described human rotavirus VP4 alleles (Wa, DS1, M37, AU228, and 69M) and used them in hybridization assays with human rotavirus strains from Latin America and Europe to determine the distribution of the VP4 gene alleles in nature. The Wa-like allele was detected most frequently, occurring in 57% of the 402 rotavirus strains tested, and the DS1-like allele was the next most common, occurring in 14% of the strains tested. The M37- and AU228-like alleles were detected in only 4 and 3% of the rotavirus strains tested, respectively, whereas the 69M-like VP4 gene allele was not detected. Several rotavirus strains from Europe did not react with any of the VP4 gene probes, although they did hybridize to a probe generated from a representative strain from the group. These data indicate the global distribution of various VP4 gene alleles and raise the possibility that other, unrecognized human VP4 alleles exist in nature because almost one-fourth of the strains could not be classified into any of the established VP4 groups.
Routine application of high-performance liquid chromatography for identification of mycobacteria
Authors: Thibert L , Lapierre S
Published: January 2020

Mycolic acid analysis by high-performance liquid chromatography (HPLC) was introduced in our laboratory as the routine technique for identifying all clinical isolates of mycobacteria referred to us. HPLC identified 96.1% of the 1,103 strains analyzed, whereas the biochemical procedures and/or the commercial DNA probes identified 98.3% of strains, for an overall agreement of 94.4%. Compared with the probes, there was 100% specificity and 98.9% sensitivity for Mycobacterium tuberculosis identification. HPLC allowed early detection and identification of the rare mycobacterial species M. haemophilum, M. malmoense, M. shimoidei, and M. fallax as well as uncharacteristic strains of M. simiae. After 18 months of routine use, HPLC proved to be reliable, easy to perform, rapid, and less costly than other identification methods.
Hepatitis C markers in hemodialysis patients
Authors: Huang C S , Ho M S , Yang C S , Lee C L , Tan C A
Published: January 2020

The prevalence of hepatitis C virus (HCV) infection among the patients of a hemodialysis unit in Taiwan was determined by an immunoblot and reverse transcriptase-polymerase chain reaction algorithm to be 58.8% (67 of 114 patients) after serological surveys with two advanced-generation enzyme-linked immunosorbent assays (ELISAs) for anti-HCV and a C 100-3 single-antigen test. The results of the second-generation ELISAs, the supplementary immunoblot test, and the test for HCV RNA were in good agreement with each other, from 86.0 to 98.2%. The first-generation C 100-3 test lacked the sensitivity of the four other systems. The two advanced-generation screening ELISAs for anti-HCV, a multiple-recombinant-antigen test, the Abbott second-generation ELISA, and a synthetic peptide multiple-antigen test, the UBI HCV EIA, provided reliable and virtually equivalent detection of potentially infected blood. Antibodies to capsid 1 and capsid 2 determinants of the Liatek immunoblot system were the most frequently detected reactivities to HCV in the HCV-infected hemodialysis patients. The percentage of HCV-infected patients with abnormal liver function (alanine aminotransferase level, greater than 100 IU/liter) was higher than that of the uninfected patients. The prevalence of HCV infection was correlated to the duration of hemodialysis treatment and the amount of blood transfused, and the most common transmission mode was thought to be patient-to-patient transmission through the dialysis equipment. Several means of reducing the frequency of transmission between hemodialysis patients are suggested.
Effects of fixation and varying target length on the sensitivity of polymerase chain reaction for detection of human T-cell leukemia virus type I proviral DNA in formalin-fixed tissue sections
Authors: Honma M , Ohara Y , Murayama H , Sako K , Iwasaki Y
Published: January 2020

In this study, the fixation condition most suitable for maintaining the sensitivity of the polymerase chain reaction (PCR) was investigated by using the alpha-tubulin gene sequence, and the PCR procedure most effective for detecting human T-cell leukemia virus type I (HTLV-I) proviral sequences in fixed, embedded tissues of adult T-cell leukemia patients was explored. First, the sensitivity of the PCR targeting a 286-bp alpha-tubulin sequence was studied in tissue sections fixed in several fixatives for various periods at 25 or 4 degrees C. For histological examination, fixation with 10% buffered formalin at a lower temperature for a shorter period was found to be preferable to retain the sensitivity. And the HTLV-I sequence was detected in only 7 of 18 specimens (38.9%) when the 374-bp sequence of the gag region was targeted, but the rate increased to 77.8% (14 of 18 specimens) when the length of the target sequence was reduced to 120 bp within the same region. Therefore, the one-round PCR targeting a shorter sequence is preferable for application of PCR to archival fixed tissue specimens, the fixation condition of which may not be ideal for DNA preservation.
Serotype analysis of rotaviruses from different locations in Malaysia
Authors: Rasool N B , Green K Y , Kapikian A Z
Published: January 2020

The distribution of rotavirus G (VP7) serotypes circulating in four locations in Malaysia, representing three geographical areas, was evaluated in 341 RNA-positive stool specimens obtained discontinuously between 1977 and 1988 from infants and young children under the age of five years who were hospitalized with acute gastroenteritis. A total of 306 specimens (256 stool suspensions and 50 that were adapted to growth in tissue culture) that were rotavirus positive by the confirmatory enzyme-linked immunosorbent assay (ELISA) were examined for serotype by ELISA utilizing monoclonal antibodies to rotavirus G serotype 1, 2, 3, 4, or 9. One hundred eighty (59%) of the 306 specimens could be serotyped; of these 180 specimens, 71% were serotype 4, 15% were serotype 1, 4% were serotype 2, and 4% were serotype 3. Serotype 9 rotavirus was not detected. Most (71%) of the specimens tested were obtained in 1988, when serotype 4 predominated in three locations in West Malaysia; no single serotype was predominant in a limited number of specimens from East Malaysia.
Antigen detection and immunological typing of Haemophilus ducreyi with a specific rabbit polyclonal serum
Authors: Roggen E L , Pansaerts R , Van Dyck E , Piot P
Published: January 2020

A rabbit polyclonal serum was raised against the 29-kDa species-specific marker, as well as the 30- to 34-kDa immunotype-specific markers of Haemophilus ducreyi described elsewhere (E. Roggen, S. De Breucker, E. Van Dyck, and P. Piot, Infect. Immun. 60:590-595, 1992). These antigens were purified from a cocktail of H. ducreyi isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immune serum reacted in enzyme-linked immunosorbent assay (ELISA) preferentially with H. ducreyi, at a titer as high as 50,000. To make it specific to H. ducreyi, nonspecific antibodies were removed by adsorption on a mixture of Haemophilus spp., Escherichia coli, Candida albicans, and Corynebacterium spp. In the 29- to 34-kDa region of immunoblot profiles from H. ducreyi isolates (n = 450), the adsorbed serum revealed essentially the same antigens as did a pool of well-characterized human sera. Yet, eight different immunotypes were observed. With this rabbit polyclonal serum, an ELISA-based antigen detection test was developed. The adsorbed serum reacted specifically with all H. ducreyi isolates tested (n = 450), but not with other bacterial species (n = 15). This test was evaluated with a limited number of clinical specimens from African patients with culture-proven chancroid and no evidence for any other ulcerating etiology (n = 10) and a number of chancroid-negative control patients from Belgium (n = 20). Within this context, the test yielded a sensitivity and specificity of 100%.
Growth and survival of Serratia marcescens under aerobic and anaerobic conditions in the presence of materials from blood bags
Authors: Szewzyk U , Szewzyk R , Stenström T A
Published: January 2020

Several patients receiving blood transfusions during the summer of 1991 developed bacteremia after the transfusion. In all cases, the infection was caused by Serratia marcescens. The same strain of Serratia marcescens was isolated from the patients and from the outer surface of unfilled blood bags. The transport containers for the blood bags were made anoxic by using a catalyst in order to prevent microbial growth. The survival and growth of S. marcescens K202, which was isolated from the blood bags, was studied at different oxygen concentrations in deionized water containing materials derived from the blood bags. The rate of survival and growth of S. marcescens was highest under anaerobic conditions, in which growth occurred with all materials and even in deionized water alone. In contrast, S. marcescens did not survive in control cultures under semi-anaerobic and aerobic conditions. Growth was observed, however, under both aerobic and semi-anaerobic conditions in the presence of each of the tested blood bag materials. These findings indicate that the conditions in the transport containers for the blood bags were favorable for the survival and growth of S. marcescens.
Identification and characterization of Lyme disease spirochetes, Borrelia burgdorferi sensu lato, isolated in Korea
Authors: Park K H , Chang W H , Schwan T G
Published: January 2020

Lyme disease spirochetes, Borrelia burgdorferi sensu lato, were identified and characterized for the first time in Korea. Four isolates, designated Konkuk-1, Konkuk-2, Kangwon-3, and KM-4, were made from midgut suspensions of three Ixodes ticks and heart tissue from one mouse, Apodemus agrarius, collected from Chungbuk and Kangwon provinces. The four Korean isolates and B. burgdorferi sensu lato from other geographic areas and biological sources were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein profiles, Western blot (immunoblot) analysis for reactivities with monoclonal and polyclonal antibodies, and agarose gel electrophoresis for plasmid profiles. Two typing schemes using polymerase chain reaction identified three of the isolates as members of group VS461 and one, Kangwon-3, as Borrelia garinii. These results demonstrate the potential for human Lyme disease to occur in some provinces of Korea.
Cloning of a DNA sequence unique to Clostridium botulinum group I by selective hybridization
Authors: McKinney M W , Levett P N , Haylock R W
Published: January 2020

Nucleic acid sequences were isolated from a strain of Clostridium botulinum type A by a selective hybridization method known as deletion enrichment. Nontoxigenic C. sporogenes was used to produce a C. botulinum type A sequence-enriched library. A probe, pCBM44, which showed specific hybridization to a 4.0-kb HindIII fragment present in all of the C. botulinum type A strains tested was isolated, and there was no hybridization to any strains of C. sporogenes. Upon further investigation, pCBM44 was found to hybridize to all of the group I proteolytic C. botulinum strains tested (toxin types A, B, and F) but not to hybridize to groups II, III, and IV (toxin types B, C, D, or E). The probe did not cross-react with nine other Clostridium spp. Such a probe, which differentiates between nontoxigenic C. sporogenes and neurotoxigenic C. botulinum group I strains, should prove extremely useful.
Development of a competitive enzyme-linked immunosorbent assay for detection of bovine, ovine, porcine, and equine antibodies to vesicular stomatitis virus
Authors: Afshar A , Shakarchi N H , Dulac G C
Published: January 2020

Two competitive (C) enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of antibodies to vesicular stomatitis virus (VSV) in animal sera. The assays are based upon the availability of polyclonal antibodies (PAbs) from mouse ascitic fluids prepared against the New Jersey (NJ) and the Indiana (IN) VSV serotypes. The assays were performed by the immobilization of VSV-NJ and VSV-IN antigens on a solid phase (microtiter plate). Appropriately diluted test serum mixed with an equal volume of serotype-specific PAb was allowed to incubate in the presence of the relevant VSV antigen and finally with an enzyme-conjugated anti-mouse immunoglobulin. In the absence of anti-VSV antibodies in the test serum, the VSV antigen sites are reactive with the relevant PAb (NJ or IN) as indicated by color development after enzyme degradation of substrate. If the test serum contains the homologous VSV-NJ or VSV-IN antibodies, they compete with the relevant PAb for immobilized antigen sites and quantitatively block and inhibit the PAb reaction and subsequent color development. The performance of C-ELISAs in detecting anti-VSV antibodies in serum samples from four calves, two horses, four sheep, and seven pigs experimentally infected with VSV-NJ and VSV-IN was evaluated. The sensitivity and specificity of the C-ELISAs were compared with those of the standard microtiter serum neutralization (MTSN) tests. Homologous antibodies were demonstrable by C-ELISAs as early as 5 days postinfection (DPI) in one horse and one sheep infected with VSV-IN serotype. Seroconversion was demonstrable by C-ELISAs and MTSN tests in all animals by 9 DPI except in one sheep that received VSV-NJ and one horse inoculated with VSV-IN serotype which, on the basis of the MTSN test results, did not seroconvert until 14 and 11 DPI, respectively. The dynamics of homologous antibody response in all animals as revealed by the corresponding type-specific C-ELISAs paralleled the results of the MTSN tests. The type-specific antibodies to VSV serotypes increased exponentially during the first 2 to 4 weeks postinfection and remained relatively stable for about 6 months in some animals. The results suggest that the C-ELISAs offer many advantages over the MTSN tests and have potential applications as rapid and inexpensive tests in serodiagnosis of VSV infections in animals.
Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis
Authors: Grenier D , Michaud J
Published: January 2020

The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity.
Levofloxacin disk potency and tentative interpretive criteria for susceptibility tests
Authors: Pfaller M A , Barry A L , Fuchs P C
Published: January 2020

Levofloxacin disk susceptibility test criteria were evaluated by testing 350 bacterial isolates. Either 5- or 10-micrograms disks could be used satisfactorily. A 5-micrograms levofloxacin disk with zone size breakpoints of < or = 12 mm for resistance (MIC, > or = 8.0 micrograms/ml) and > or = 16 mm for susceptibility (MIC, < or = 2.0 micrograms/ml) is recommended.
Ramoplanin susceptibility testing criteria
Authors: Barry A L , Pfaller M A , Fuchs P C
Published: January 2020

Ramoplanin and mupirocin are two antimicrobial agents that may be applied topically. They had similar potencies against susceptible gram-positive cocci, but they differed in their spectra of activity. In plastic microdilution trays, the in vitro activity of ramoplanin was slightly diminished but that activity was restored by the addition of bovine serum albumin (0.02%). Susceptible strains were defined as those for which MICs of ramoplanin were < or = 2.0 micrograms/ml (without bovine serum albumin) and which had > or = 11-mm zones around 60-micrograms ramoplanin disks. Criteria for a resistant category cannot be defined at this time.
Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1
Authors: Gomez-Lus P , Fields B S , Benson R F , Martin W T , O'Connor S P , Black C M
Published: January 2020

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to characterize Legionella pneumophila serogroup 1. Cells from a single colony could be subtyped by AP-PCR within a few hours. The discrimination between strains of L. pneumophila serogroup 1 by AP-PCR was equivalent to that by monoclonal antibody analysis and ribotyping. Four strains representing the monoclonal antibody pattern most frequently associated with outbreaks all yielded unique amplicon patterns by AP-PCR.
Cell-free plasma human immunodeficiency virus type 1 titer assessed by culture and immunocapture-reverse transcription-polymerase chain reaction
Authors: Coombs R W , Henrard D R , Mehaffey W F , Gibson J , Eggert E , Quinn T C , Phillips J
Published: January 2020

The relationship between plasma human immunodeficiency virus type 1 (HIV-1) infectious titer, determined by quantitative fivefold end-point dilution culture, and the detection of genomic HIV-1 RNA by immunocapture-cDNA-polymerase chain reaction was determined. The optimal plasma specimen collection and storage conditions for the use of such virologic markers for clinical trials were also determined. The variabilities in the measurement of infectious HIVLAI titer associated with intra- and interdonor peripheral blood mononuclear cells were 1.2 and 0.86 log10 50% tissue culture infective doses (TCID50)/ml (95% confidence interval range), respectively. Plasma HIV-1 titers did not change significantly after storing whole blood for 6 h either at 4 degrees C or ambient temperature or plasma for a median of 267 days (range, 259 to 482) at -70 degrees C. The detection of genomic HIV-1 RNA encapsulated in viral particles was very consistent, reproducible, and unaffected by either heparin or acid citrate or by multiple freeze-thawing. The HIV-1 RNA titers also appeared to generally correlate with the biologic titer obtained by the microculture assay. The consistency in infectious HIV-1 titer was evaluated by using 27 unfrozen plasma specimens collected from five subjects over 1 to 9 days. The median change in HIV-1 titer relative to baseline was -0.5 log10 TCID50/ml (interquartile range, -1.03 to 0.175 log10). In contrast, no significant change in HIV-1 RNA for the same frozen plasma specimens was noted. As such, immunocapture-cDNA-polymerase chain reaction may be a useful measure of plasma viremia for studying the natural history of HIV disease and assessing response to therapy.
Comparative evaluation of AccuProbe culture identification test for Neisseria gonorrhoeae and other rapid methods
Authors: Young H , Moyes A
Published: January 2020

The AccuProbe chemiluminescent culture identification test for Neisseria gonorrhoeae (Gen-Probe Inc., San Diego, Calif.) was assessed in a comparative evaluation with other rapid methods by using 269 isolates of oxidase-positive, gram-negative diplococci. Chemiluminescence was read with a PAL luminometer, and results were expressed as PAL light units (PLUs): the cutoff value for a positive identification was 1,500 PLUs. All 200 isolates of gonococci confirmed by carbohydrate utilization and serotyped with monoclonal antibodies were identified correctly by AccuProbe on initial testing. The API Quadferm system (Bio Merieux, Marcy l'Etoile, France) identified 95% (n = 190) of the gonococci correctly on initial testing and 99.5% (n = 199) on repeat testing, while the Phadebact Monoclonal GC test (Kara Bio Diagnostics AB, Huddinge, Sweden) identified 95.5% (n = 191) of the gonococci on both initial and repeat testing; 8 of the Phadebact-negative isolates were all of the same rare serovar (serovar 1B-17). The mean PLU for the gonococcal isolates was 9,014 (range 2,264 to 10,845) compared with a mean of 51 (range, 8 to 109) for the 69 nongonococcal isolates. We conclude that the AccuProbe culture confirmation test provides a rapid and accurate objective means of identifying cultured N. gonorrhoeae isolates.
Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis
Authors: Vaneechoutte M , De Beenhouwer H , Claeys G , Verschraegen G , De Rouck A , Paepe N , Elaichouni A , Portaels F
Published: January 2020

A rapid procedure for the identification of cultured Mycobacterium isolates, based on the combination of enzymatic amplification and restriction analysis, is described. The 16S rRNA genes (rDNA) of 99 strains belonging to 18 different species of the genus Mycobacterium were enzymatically amplified. Amplified rDNA restriction analysis with the enzymes CfoI, MboI, and RsaI was carried out. The combination of the amplified rDNA restriction analysis patterns obtained after restriction with CfoI and MboI enabled differentiation between Mycobacterium asiaticum (number of strains = 4), M. avium (n = 22), M. chelonae (n = 5), M. flavescens (n = 1), M. fortuitum (n = 6), M. gordonae (n = 6), M. intracellulare (n = 13), M. marinum (n = 7), M. nonchromogenicum (n = 1), M. simiae (n = 5), M. terrae (n = 5), the M. tuberculosis complex (n = 11), and 2 of 4 strains of M. xenopi. Further restriction with RsaI was necessary to differentiate between the species M. kansasii (n = 5), M. scrofulaceum (n = 4), and the 2 other M. xenopi strains. The M. avium-M. intracellulare complex was characterized by a specific MboI pattern, and M. avium and M. intracellulare strains could further be differentiated by restriction with CfoI. The whole procedure, including sample preparation prior to the polymerase chain reaction, can be carried out within 8 h, starting from a pure culture.
Diagnostic value of amplification of human cytomegalovirus DNA from gastrointestinal biopsies from human immunodeficiency virus-infected patients
Authors: Cotte L , Drouet E , Bissuel F , Denoyel G A , Trepo C
Published: January 2020

In order to assess the value of human cytomegalovirus (HCMV) DNA amplification of gastrointestinal biopsies, we studied 57 human immunodeficiency virus-infected patients with and without gastrointestinal HCMV diseases. After DNA extraction, a 406-bp fragment from the unique short region of the HCMV genome was amplified by 35 cycles of polymerase chain reaction (PCR) and semiquantified from 80 to 80,000 HCMV genomic copies. Among 12 non-AIDS patients, the PCR assay was negative for 11 of 12 duodenal and 8 of 8 colorectal samples. It was also negative for 28 of 31 duodenal and 12 of 15 colorectal samples from 31 AIDS patients without gastrointestinal HCMV diseases. Among 14 AIDS patients with gastrointestinal HCMV diseases, the PCR assay was positive for 12 of 12 patients with HCMV duodenitis and for 13 of 13 patients with HCMV colitis. Results were dichotomized between high and low HCMV-DNA copy numbers. For duodenitis, sensitivity was 92% and specificity was 100%. For colitis, sensitivity was 92% and specificity was 93%. Specificity and sensitivity were not influenced by shedding status for HCMV or by other gastrointestinal infections. HCMV DNA amplification of gastrointestinal biopsies is a sensitive and specific tool for the diagnosis of gastrointestinal HCMV diseases in AIDS patients.
Identification of an insertion sequence-like element in a subspecies of Mycobacterium kansasii
Authors: Yang M , Ross B C , Dwyer B
Published: January 2020

Analysis of a genomic DNA clone library of a strain from the genetic subspecies of Mycobacterium kansasii determined the existence of a repetitive insertion sequence-like element. The element is 947 bp long and is present in a minimum of 1 to 11 copies per genome. Similar to insertion sequences, it contains a 3-bp (TAG) direct repeat at its extremities and a transcription promoter-like sequence. In addition, for one of the clones sequenced, a potential cointegrate formation, a characteristic frequently observed with insertion sequences, was revealed. This insertion sequence does not contain short inverted repeats near the ends or a large open reading frame to code for a transposase enzyme. Its host range is restricted to a previously described genetic subspecies of M. kansasii and is not present in typical M. kansasii or other mycobacterial species. When used as a probe for Southern blot hybridization, significant heterogeneity between different isolates of the M. kansasii subspecies was observed. This repeated element will be useful in further studies on the characterization, diagnosis, and epidemiology of M. kansasii.
Rationale for selective use of anaerobic blood cultures
Authors: Morris A J , Wilson M L , Mirrett S , Reller L B
Published: January 2020

Because of the declining frequency of anaerobic bacteremia, routinely using half the collected blood volume for anaerobic culture has been challenged. There is no data indicating whether more clinically relevant isolates would be recovered if all or most of the given blood sample were cultured aerobically. In this two-part study, we reviewed cases of anaerobic bacteremia to determine what proportion occurred in situations when anaerobes would be expected and then estimated the yield of different culture approaches by reanalyzing the data from a large prospective clinical blood culture study. The records of 61 patients who had an anaerobic isolate (excluding Propionibacterium species) recovered only from an anaerobic bottle were examined to define clinical settings in which such isolates occur. Fifty-six (92%) patients had clinically important isolates, and the source of infection was obvious at the time of culture in 47 of the 56 (84%). Of 56 patients, 36 (64%) had abdominal signs and symptoms, including 12 with recent abdominal surgery. Of nine patients without an obvious source of infection, six were on high-dose steroids. Relative yields were compared for (i) one aerobic bottle and one anaerobic bottle (5 ml to each) for all blood cultures, (ii) two aerobic bottles (5 ml to each), or (iii) two aerobic bottles plus an extra anaerobic bottle (only for clinically suspected anaerobic sepsis) (5 ml to each). The third approach had the highest yield (475 isolates), because the routine use of two aerobic bottles recovered more Candida spp., members of the family Enterobacteriaceae, and nonfermenters than did the first approach (448 isolates) (P < 0.02), and clinically directed culturing for anaerobes would recover anaerobes missed with the second approach (458 isolates). Our data suggest that the use of two aerobic bottles with selective culturing for anaerobes could increase the number of clinically relevant isolates by at least 6% compared with the current practice of inoculating an aerobic bottle and an anaerobic bottle with equal volumes of blood.
Oligonucleotide fingerprinting of isolates of Candida species other than C. albicans and of atypical Candida species from human immunodeficiency virus-positive and AIDS patients
Authors: Sullivan D , Bennett D , Henman M , Harwood P , Flint S , Mulcahy F , Shanley D , Coleman D
Published: January 2020

Oligonucleotide fingerprinting of genomic DNA from oral isolates of four different Candida species other than C. albicans and atypical chlamydospore-positive isolates from human immunodeficiency virus (HIV)-positive individuals and AIDS patients was investigated as a means for differentiating between isolates within individual species. Oligonucleotides composed of simple repetitive sequence motifs, including (GACA)4, (GATA)4, (GGAT)4, (GTG)5, and (GT)8, all yielded fingerprints suitable for strain segregation of 8 C. tropicalis isolates, 12 Torulopsis (Candida) glabrata isolates, 8 atypical Candida isolates, and, except for (GATA)4, 2 C. krusei probe in turn and so generate several distinct DNA fingerprints of the same DNA sample. However, none of the probes yielded fingerprints suitable for strain segregation with three C. parapsilosis isolates. The (GATA)4 probe was also used to detect restriction fragment length polymorphisms among a genetically closely related group of atypical Candida isolates on primary isolation from an additional HIV-infected patient. These chlamydospore-positive atypical Candida isolates were sucrose positive, were of C. albicans serotype A, hybridized weakly with the C. albicans-specific mid-repeat sequence probe 27A, and yielded fingerprint profiles by random polymorphic DNA analysis that were distinct from those derived from C. albicans isolates. The C. stellatoidea ex-type strain NCPF 3108 was indistinguishable from the atypical Candida isolates in all these tests and also yielded an identical carbohydrate and nitrogen source assimilation profile by using the ID 32C yeast identification system.
Evaluation of the molecular epidemiology of an outbreak of multiply resistant Shigella sonnei in a day-care center by using pulsed-field gel electrophoresis and plasmid DNA analysis
Authors: Brian M J , Van R , Townsend I , Murray B E , Cleary T G , Pickering L K
Published: January 2020

Outbreaks of diarrhea in child day-care centers (DCC) are common. This study was undertaken to evaluate the molecular epidemiology of an outbreak of diarrhea due to Shigella sonnei. This outbreak involved 25 of 52 (48%) DCC children and 14 of 132 (11%) teachers and household contacts. S. sonnei isolates from nine children and five contacts were characterized by antimicrobial susceptibility, plasmid content, plasmid DNA restriction fragment pattern, and pulsed-field gel electrophoresis (PFGE) of total genomic DNA; 33 isolates from Houston, Tex., Chicago, Ill., and Mexico City, Mexico, also were studied. All outbreak isolates were resistant to ampicillin and trimethoprim-sulfamethoxazole and shared five to six plasmids ranging from 3.3 to 70 MDa. A total of 8 of 12 temporally associated nonoutbreak Houston isolates had plasmid profiles and restriction fragment patterns similar to those of the outbreak strain, despite possessing different antibiotic susceptibility patterns. PFGE demonstrated identical DNA patterns among outbreak isolates and similar or identical patterns among temporally associated sporadic Houston isolates with plasmid profiles similar to that of the outbreak strain. All other nonoutbreak strains from Houston, Chicago, and Mexico had plasmid profiles, restriction fragment patterns, and PFGE patterns different from those of the outbreak strain. DCC outbreak isolates could be distinguished from most sporadic isolates by antimicrobial susceptibility testing, but plasmid analysis and PFGE could not differentiate common-source isolates from sporadic isolates in the same location during the same time period, indicating that isolates present in the community were genetically similar to those producing outbreaks in the DCC.
Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli
Authors: Ghosh A R , Sen D , Sack D A , Hoque A T
Published: January 2020

Strains of enterotoxigenic Escherichia coli producing either colonization factor antigen (CFA) I or II were tested for expression of CFA when grown on 16 different agar media by using agglutination and coagglutination with monoclonal antibodies, mannose-resistant hemagglutination, and a salt aggregation assay. CFA was detected from the CFA-positive strains when CFA agar was used, and it was also detected when other commercially available media were used, notably nutrient agar. CFA was not detected when other commercial media such as MacConkey agar were used. The use of nutrient agar with monoclonal antibody-based coagglutination reagents offers a potentially simple and rapid method for detecting E. coli which express CFA I or II.
Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis
Authors: Gottschalk M , Higgins R , Boudreau M
Published: January 2020

Polyvalent coagglutination reagents (PRs) have been evaluated for the serotyping of Streptococcus suis. Monovalent antisera produced against 28 S. suis reference strains have been grouped to obtain five different pools. A total of 249 field isolates previously identified and belonging to different serotypes were tested with PRs prepared by two different procedures: (i) monovalent coagglutination reagents were individually prepared and mixed in equal proportions, and (ii) antisera were mixed in equal proportions before the addition of the Staphylococcus aureus suspension. Only antisera tested by a tube agglutination test with 2-mercaptoethanol and presenting titers of 1:32 or higher were used. Results obtained with PRs prepared by both procedures were similar, and there was a very good correlation between the capsular type of the isolate and the reaction obtained with PRs. Thus, from a practical viewpoint, it is suggested that PRs be prepared by the first procedure. To isolates, were tested in parallel with both the PRs and the monovalent coagglutination reagents over a 1-year period. Ninety-nine percent of the typeable and all of the untypeable isolates were correctly identified. Serotyping with PRs is suggested to be a very useful and reliable screening procedure, particularly when a large number of S. suis isolates have to be serotyped. In addition, the choice of antisera to be included in a given pool is facultative and should be oriented to the needs of a region or a country.
Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays
Authors: deWit D , Wootton M , Allan B , Steyn L
Published: January 2020

A simple method for the production of internal control DNA for two well-established Mycobacterium tuberculosis polymerase chain reaction assays is described. The internal controls were produced from Mycobacterium kansasii DNA with the same primers but at a lower annealing temperature than that used in the standard assays. In both assays, therefore, the internal control DNA has the same primer-binding sequences at the target DNA. One-microgram quantities of internal control DNA which was not contaminated with target DNA could easily be produced by this method. The inclusion of the internal control in the reaction mixture did not affect the efficiency of amplification of the target DNA. The method is simple and rapid and should be adaptable to most M. tuberculosis polymerase chain reaction assays.
Tentative interpretive criteria for disk diffusion susceptibility testing of sparfloxacin
Authors: Fuchs P C , Barry A L , Pfaller M A
Published: January 2020

Susceptibility to sparfloxacin was tested simultaneously by the agar dilution and disk diffusion methods. For tests with 5-micrograms sparfloxacin disks, we propose that the breakpoints for the susceptibility and resistance categories should be > or = 19 and < or = 15 mm, respectively. Most minor discrepancies involved tests with pseudomonads.
Construction of a bioluminescent mycobacterium and its use for assay of antimycobacterial agents
Authors: Andrew P W , Roberts I S
Published: January 2020

To show, as a model system, that mycobacteria can express heterologous luciferase genes and that bioluminescence can be a rapid method of measuring antimycobacterial activity, a bioluminescent form of Mycobacterium smegmatis was made by transformation with a Mycobacterium-Escherichia coli shuttle vector containing the luxAB genes from Vibrio harveyi. The antimycobacterial effects of antibiotics and biocides could be assayed in real time by using bioluminescent M. smegmatis.
DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae
Authors: Doran J L , Collinson S K , Burian J , Sarlós G , Todd E C , Munro C K , Kay C M , Banser P A , Peterkin P I , Kay W W
Published: January 2020

Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp.
Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans
Authors: Meyer W , Mitchell T G , Freedman E Z , Vilgalys R
Published: January 2020

In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms.