bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity, persistence, and distribution of Pseudomonas aeruginosa genotypes in cystic fibrosis patients
Authors: Fegan M , Francis P , Hayward A C , Fuerst J A
Published: January 2020

A collection of 222 isolates of Pseudomonas aeruginosa was obtained from the respiratory tract of 16 patients with cystic fibrosis over a 4- to 9-month period. Fourteen of these patients were unrelated, while the remaining two were siblings. Isolates were typed by conventional pyocin typing and also by the use of a DNA probe containing 741 bp immediately upstream of the exotoxin A structural gene and the initial 732 bp of the exotoxin A structural gene. By pyocin typing, 69% (11 of 16) of the patients were shown to harbor a single type that persisted in the lung throughout the study. By genotyping (DNA probe typing), all but three patients (13 of 16, 81%) harbored a single persistent genotype in their lungs. Six patients other than the sibling pair (6 of 14, 43%) shared a common genotype in their lungs as judged by DNA probing, and the pyocin type of these isolates was also identical. In four of these six patients, the shared genotype was also the persistent genotype. The sibling pair studied also carried a common genotype in their lungs as indicated by DNA probing, even though the pyocin type of these isolates varied. Results presented suggest that the majority of patients harbor a persistent strain in their lungs and that cross-colonization may occur.
Use of synthetic oligonucleotide DNA probes for identification and direct detection of Bacteroides forsythus in plaque samples
Authors: Moncla B J , Motley S T , Braham P , Ewing L , Adams T H , Vermeulen N M
Published: January 2020

Oligonucleotide DNA probes complementary to the hypervariable region of the 16S rRNA of Bacteroides forsythus were tested for their specificity and sensitivity against reference and clinical isolates of 73 different species of bacteria. The probes were used to detect the organism directly from nucleic acid extracts of subgingival plaque samples, and the results were compared with results of detection by culture methods. The data demonstrate that the probes are very specific (100%) and sensitive (100% when they are compared with those obtained by the culture method) and could reliably detect the organism in plaque samples. When a probe to a conserved region of the 16S rRNA (UP9A) was used to estimate the quantity of bacteria present in plaque samples, it gave results comparable to those of culture methods. The data suggest that total microbial load may be a parameter in periodontal disease.
Identification of two new antigenic subgroups within the genus Mobiluncus
Authors: Schwebke J R , Lukehart S A , Roberts M C , Hillier S L
Published: January 2020

Classifications of 48 atypical clinical isolates of Mobiluncus spp. were determined on the basis of biochemical reactions, morphology, antigenic profiles, and DNA hybridization studies. Two new subgroups with unique antigenic profiles are described. Like typical Mobiluncus species, the antigenic variant of M. mulieris is associated with bacterial vaginosis. The atypical isolates of M. curtisii were frequently recovered from women with normal vaginal flora and were also recovered from sterile body sites. These isolates may be incorrectly identified if current biochemical and morphological criteria are used for identification. Gram stain morphology, however, correctly identifies these isolates to the species level. The characterization of these atypical isolates has important implications for future investigations in which serological methods are used for diagnosis, epidemiology, and determination of pathogenicity of Mobiluncus spp.
Epidemiological survey of human rotavirus serotypes and electropherotypes in young children admitted to two children's hospitals in northeast London from 1984 to 1990
Authors: Noel J S , Beards G M , Cubitt W D
Published: January 2020

A retrospective and prospective survey was carried out to determine the relative frequency of rotavirus serotypes infecting children with diarrhea or vomiting or both who were admitted to the Hospitals for Sick Children in London during a 6-year period from 1984 to 1990. The results were compared with data for the same period from a study in Birmingham, United Kingdom. The serotype of rotaviruses infecting 1,019 children was ascertained by enzyme immunoassay with VP7-specific monoclonal antibodies. In London, serotype G1 accounted for 60% of the cases, serotype G4 accounted for 24%, serotype G2 accounted for 11%, G3 accounted for 3%, and coinfections accounted for 2%. Considerable differences in the relative prevalence of serotypes were seen when data from London and Birmingham were compared. A major shift from serotype G1 to G4 was observed in London in the 1989 to 1990 season, and a lesser shift was seen in Birmingham. Examination of the electrophoretic profiles of 611 rotaviruses from London showed that there were at least 108 different profiles. Continuous variation occurred throughout the 6-year period, and the same electropherotype never recurred once it had disappeared from the population. None of the electrophoretic profiles were characteristic of group B or group C rotaviruses. There was no evidence that any strain of rotavirus had become endemic in either of the children's hospitals in London.
Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction
Authors: Murakami K , Minamide W , Wada K , Nakamura E , Teraoka H , Watanabe S
Published: January 2020

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.
Clinical comparison of the resin-containing BACTEC 26 Plus and the Isolator 10 blood culturing systems
Authors: Tarrand J J , Guillot C , Wenglar M , Jackson J , Lajeunesse J D , Rolston K V
Published: January 2020

The new resin-containing BACTEC 26 Plus blood culturing system (Becton Dickinson Diagnostic Instrument Systems, Towson, Md.) was compared with the Isolator 10 system (Wampole Laboratories, Cranbury, N.J.). Blood samples were drawn by syringe, and equal 10-ml volumes were evaluated in each blood culture system by the recommended methods. Both systems were incubated aerobically with 5% CO2. Of 11,506 acceptable study specimens, 1,788 aerobic isolates were recovered. Overall, recoveries was similar for the two systems, with 626 bacteria or fungi recovered in the BACTEC 26 Plus system only, 499 recovered in the Isolator system only, and 663 recovered in both systems. Of 345 gram-negative rods, 62 grew in the BACTEC system only and 109 grew in the Isolator system only (P less than 0.001). Thirty-three of these Isolator-only gram-negative organisms were Acinetobacter spp. Of 209 yeasts, 38 grew in BACTEC only and 81 grew in Isolator only (P less than 0.001). Of 200 streptococci and enterococci, 98 were recovered in BACTEC only and 26 grew in Isolator only (P less than 0.001). Two hundred twenty-eight independent episodes of gram-negative rod bacteremia occurred. Isolator was the first system positive in 59 of 197 episodes, compared with 45 of 197 for BACTEC when Acinetobacter episodes were excluded. Times to detection were similar for the two systems. High colony counts correlated with repeat positive blood cultures. Isolator and BACTEC had similar overall recoveries, with individual merits and deficiencies for both systems. The additional quantitative information derived from Isolator had utility in our institution.
Evaluation of the MicroScan Rapid Yeast Identification panel
Authors: St Germain G , Beauchesne D
Published: January 2020

The MicroScan Rapid Yeast Identification (RYI) panel is a 4-h microdilution system for identification of clinical yeastlike isolates. Its accuracy was evaluated by using 357 isolates encompassing 11 genera and 30 species. The RYI panel identifications were compared with those obtained by the API 20C system assisted with morphological characterization on cornmeal-Tween 80 agar. The panels were read both visually and with the AutoScan-4, a computer-controlled microplate reader. Both the RYI panel and the API 20C system correctly identified 78% of the strains within 4 and 72 h, respectively, with no additional tests. Supplementary tests recommended by the manufacturers made it possible to identify up to 96.6% (AutoScan-4) and 98.9% (API 20C) of the strains. The accuracy of the RYI panel was 99.5% with common strains and 92.1% with less common strains. The RYI panel misidentified 10 or 12 strains and failed to identify 2 or 3 strains, depending on whether it was read with the AutoScan-4 or visually. Errors occurred with one strain of Torulopsis glabrata and the less common yeasts T. candida, Candida lusitaniae, C. lambica, C. rugosa, C. stellatoidea, Cryptococcus albidus, C. laurentii, and C. uniguttulatus. Overall, the RYI panel appears to be a reliable system for identification of the more common clinical yeast isolates.
Enterosistem 18-R: description and comparative evaluation with conventional methods for identification of members of the family Enterobacteriaceae
Authors: Piccolomini R , Di Girolamo A , Catamo G , Cellini L , Allocati N , Ravagnan G
Published: January 2020

The efficiency and accuracy of Enterosistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 360 members (38 species) of the family Enterobacteriaceae. Overall, 329 strains (91.3%) were correctly identified (percentage of identification, greater than or equal to 90.0), with 37 (11.2%) requiring additional tests for complete identification. For 11 isolates (3.1%), Enterosistem 18-R gave only genus identifications, and for 14 (3.9%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only six isolates (1.7%) were misidentified. The new system accurately identified common and several newly described isolates of the family Enterobacteriaceae, such as Enterobacter gergoviae, Providencia rustigianii, Serratia odorifera, and Serratia rubidaea. The system is highly reproducible, simple to perform, easy to handle, and inexpensive. With adjustments in supplementary code numbers for some strains, Enterosistem 18-R is a suitable alternative for identification of members of the Enterobacteriaceae in clinical laboratories.
Glycoinositol phospholipids from American Leishmania and Trypanosoma spp: partial characterization of the glycan cores and the human humoral immune response to them
Authors: Avila J L , Rojas M , Acosta A
Published: January 2020

The glycoinositol phospholipid (GIPL) profiles of American Leishmania spp. (L. mexicana and L. braziliensis), Leishmania donovani, and American Trypanosoma spp. (T. cruzi and T. rangeli) were compared. The major GIPLs in these parasites include tetraglycosyl-, pentaglycosyl-, and hexaglycosylphosphatidylinositol. These were partially identified by their comigration by high-performance thin-layer chromatography with purified L. major GIPLs, gas-liquid chromatography of the monosaccharides released after aqueous HF treatment, N-acetylation and methanolysis, sensitivity to exoglycosidases, and antibody absorption on several specific natural haptens. Members of the genus Leishmania have two other highly polar glycolipids, while the T. rangeli glycolipid profile was quite different from those of other kinetoplastids that were studied. On a weight basis, the glycan core of L. major GIPL-1 is the most reactive, followed by GIPL-3 and GIPL-2. Antibodies to the core glycans of GIPL-1, GIPL-2, and GIPL-3 were present at a low titer in the serum of every normal individual studied, while elevated GIPL-2 antibody levels were present in 80 to 100% of T. cruzi-, T. rangeli-, or L. donovani-infected patients, with lower values being found for GIPL-3 (30 to 60%) and GIPL-1 (30 to 50%). Except for GIPL-2 antibodies, which were mainly located on immunoglobulin G (IgG) and IgM, GIPL-1 and GIPL-3 antibodies were mainly distributed in IgM, with lower reactivity present in IgG. Antigen-antibody binding was very selectively blocked with Gal(alpha 1-3)Man, or Gal(beta 1-4)Man, Gal(alpha 1-3)Gal, and Gal(alpha 1-6)Gal for GIPL-1, GIPL-2, and GIPL-3 antibodies, respectively.
Superiority of methylprednisolone over dexamethasone for induction of Pneumocystis carinii infection in rats
Authors: Sukura A , Soveri T , Lindberg L A
Published: January 2020

Because of difficulties in in vitro cultivation, the basic Pneumocystis carinii studies have been carried out on animal models, mainly on rodents immunosuppressed by corticosteroids. Commonly used dexamethasone and methylprednisolone procedures were evaluated. The intensity of infection in rats was statistically significantly higher after 9 weeks' immunosuppression with methylprednisolone than with dexamethasone.
Convenient agarose medium for simultaneous determination of the low-calcium response and Congo red binding by virulent strains of Yersinia enterocolitica
Authors: Bhaduri S , Turner-Jones C , Lachica R V
Published: January 2020

A simple, efficient method for identification and differentiation of Yersinia enterocolitica containing virulent plasmid-bearing clones is described. The method is based on temperature of incubation and the low calcium response of the organism in an agarose Congo red medium.
Molecular epidemiology of Yersinia enterocolitica O:3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes
Authors: Blumberg H M , Kiehlbauch J A , Wachsmuth I K
Published: January 2020

Yersinia enterocolitica is a major enteric pathogen associated with a wide variety of clinical and immunologic manifestations, including transfusion-associated disease, from which there is a high mortality. Although previously rare in the United States, in the late 1980s Y. enterocolitica O:3 emerged as the predominant serotype in the United States, as it has been in Canada, Europe, and Japan. Epidemiologic investigation of this serogroup has been hampered by the limited availability of a phage typing system and the fact that Y. enterocolitica harbors few plasmids that are useful as strain markers. We therefore analyzed whole-cell DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to study a group of 61 (50 human, 11 porcine) Y. enterocolitica isolates. Initially, 20 different restriction enzymes were used: NciI appeared to give the best discrimination of hybridization banding patterns (ribotypes) within Y. enterocolitica O:3. Ribotyping distinguished seven clones among all the study isolates and four clones within Y. enterocolitica O:3 (53 isolates studied) and clearly differentiated Y. enterocolitica O:3 from Y. enterocolitica O:9; O:1,2,3; O:20; and O:5,27. Most serogroup O:3 isolates belonged to two clones, ribotypes I and II, including 23 of 24 Y. enterocolitica O:3 (13 human, 11 porcine chitterling) isolates recovered from a recent outbreak of Y. enterocolitica in children in Atlanta associated with chitterling preparation and 3 transfusion-associated O:3 isolates from the United States. Y. enterocolitica O:3 ribotypes I and II were also isolated in Japan, ribotypes II and IV were isolated in Belgium, and ribotype I was isolated in Canada. Ribotype patterns I and II corresponded to phage types 9b and 8, respectively. Ribotyping was able to distinguish individual strains of Y. enterocolitica O:3, but suggests that a limited number of clones have disseminated within the United States and globally. The finding of identical ribotype patterns in chitterling and human specimens from the Atlanta outbreak supports epidemiologic evidence that swine were the source of infection and major reservoir for Y. enterocolitica O:3.
Rapid isolation and presumptive diagnosis of uropathogens by using membrane filtration and differential media
Authors: Friedman M P , Danielski J M , Day T E , Dunne J C , Evangelista A T , Freeman T R
Published: January 2020

Random urine samples from hospitalized patients (n = 550) and seeded sterile filtered urine samples (n = 730) were used to test a membrane filtration technique, Qualture (Future Medical Technologies International, Inc., West Palm Beach, Fla.), for the detection and identification of uropathogens. Results for each sample were compared with those obtained by the calibrated loop (0.01 ml) method to demonstrate the sensitivity of the method as a screening tool and the specificity of the presumptive diagnosis obtained from the pattern of growth on differential media. The medium was supplied as dehydrated nutrient pads (Sartorius AG, Goettingen, Germany) and was activated by rehydration by the addition of the liquid specimen. With a threshold of 10(4) CFU/ml defining a positive culture, the sensitivity of the Qualture was 100%. At lower levels of bacteriuria, the Qualture was more sensitive than the calibrated loop method. Significant infections were presumptively diagnosed at 4 h by filtration rather than at 24 h on agar medium. The specificity of uropathogen identification ranged from 99% for Enterococcus spp. to 83% for Pseudomonas spp. Citrobacter spp. could not be differentiated from Escherichia coli and Providencia spp. could not be differentiated from Proteus spp., which does not create a therapeutic dilemma. Filtration, isolation, quantitation, and presumptive diagnosis are performed in one step, without subculture. Membrane filtration is a sensitive and rapid technique, with the advantage that it can be used as a collection and transport device without the use of growth inhibitors.
Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe
Authors: Moe C L , Allen J R , Monroe S S , Gary H E , Humphrey C D , Herrmann J E , Blacklow N R , Carcamo C , Koch M , Kim K H
Published: January 2020

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.
Amplification of Borrelia burgdorferi DNA in skin biopsies from patients with Lyme disease
Authors: Melchers W , Meis J , Rosa P , Claas E , Nohlmans L , Koopman R , Horrevorts A , Galama J
Published: January 2020

To determine whether the polymerase chain reaction could contribute to a better diagnosis of Lyme disease, skin biopsy samples from patients suffering from erythema chronicum migrans or acrodermatitis chronica atrophicans were tested for the presence of Borrelia burgdorferi by a polymerase chain reaction assay, which was specific for European strains. The spirochete could not be detected microscopically in any of the 15 biopsy samples obtained from nine patients. However, B. burgdorferi could be isolated from seven of eight of these samples, which indicated the presence of spirochetes. Using a nested polymerase chain reaction, we were able to detect B. burgdorferi-specific sequences in 12 of the 15 biopsy samples. Biopsy samples from three of four patients with erythema chronicum migrans and four of five patients with acrodermatitis chronica atrophicans were found to be positive for B. burgdorferi. The spirochete could be isolated from the biopsy sample, from a patient with erythema chronicum migrans who tested negative, which suggests a false-negative polymerase chain reaction result probably on account of the low number of spirochetes present in the lesion. The positive polymerase chain reaction for lesions from patients with acrodermatis chronica atrophicans supports the concept that B. burgdorferi can persist in the skin over a long period of time. From these results, it was concluded that the polymerase chain reaction is a valuable technique for the diagnosis of Lyme disease.
Use of a urine enzyme immunoassay as a diagnostic tool for Chlamydia trachomatis urethritis in men
Authors: Schwebke J R , Clark A M , Pettinger M B , Nsubga P , Stamm W E
Published: January 2020

We collected first-voided urine specimens from 659 males attending a sexually transmitted disease clinic and performed both enzyme immunoassay (EIA) for detection of chlamydial antigen and leukocyte esterase testing on these urine samples. The overall prevalence of chlamydial urethritis in the study population as determined by culture of urethral swabs was 11%. However, 46% of all men in the study had no symptoms of urethritis. Compared with urethral cultures for chlamydiae, the urine EIA had a sensitivity of 42% and a specificity of 99%. The sensitivity of the EIA strongly correlated with the amount of antigen present in culture as assessed by numbers of inclusion-forming units. The sensitivity of the leukocyte esterase test compared with that of chlamydia culture was 88%. We conclude that in this population of men, which included many patients without symptoms of urethritis, the urine EIA was a relatively insensitive means of screening for chlamydial infection.
Proposal of Afipia gen. nov., with Afipia felis sp. nov. (formerly the cat scratch disease bacillus), Afipia clevelandensis sp. nov. (formerly the Cleveland Clinic Foundation strain), Afipia broomeae sp. nov., and three unnamed genospecies
Authors: Brenner D J , Hollis D G , Moss C W , English C K , Hall G S , Vincent J , Radosevic J , Birkness K A , Bibb W F , Quinn F D
Published: January 2020

On the basis of phenotypic characterization and DNA relatedness determinations, the genus Afipia gen. nov., which contains six species, is described. The type species is Afipia felis sp. nov. (the cat scratch disease bacillus). Afipia clevelandensis sp. nov., Afipia broomeae sp. nov., and three unnamed not associated with cat-borne disease. All but one strain (Afipia genospecies 3) were isolated from human wound and respiratory sources. All Afipia species are gram-negative, oxidase-positive, nonfermentative rods in the alpha-2 subgroup of the class Proteobacteria. They are motile by means of a single flagellum. They grow on buffered charcoal-yeast extract agar and nutrient broth, but rarely on MacConkey agar, at 25 and 30 degrees C. They are urease positive; but they are negative in reactions for hemolysis, indole production, H2S production (triple sugar iron agar), gelatin hydrolysis, esculin hydrolysis, and peptonization of litmus milk. They do not produce acid oxidatively from D-glucose, lactose, maltose, or sucrose. The major cell wall fatty acids are 11-methyloctadec-12-enoic (CBr19:1), cis-octadec-11-enoic (C18:1omega7c), and generally, 9,10-methylenehexadecanote and 11,12-methyleneoctadecanoate; and there are only trace amounts of hydroxy acids. The guanineplus-cytosine content is 61.5 to 69 mol%. A. felis is positive for nitrate reduction and is delayed positive for acid production from D-xylose, but it is catalase negative. A. clevelandensis is negative in all of these tests. A. broomeae is weakly positive for catalase production and acid production from D-xylose, but it is negative for nitrate reduction.
Rapid detection of type A influenza viruses with monoclonal antibodies to the M protein (M1) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay
Authors: Bucher D J , Mikhail A , Popple S , Graves P , Meiklejohn G , Hodes D S , Johansson K , Halonen P E
Published: January 2020

Monoclonal antibodies (MAbs) to the M protein (M1) were used in the development of direct detection systems for type A influenza viruses in clinical specimens. Optimal detection by an enzyme-linked immunosorbent assay was achieved when MAbs were used as capture antibodies and rabbit polyclonal antibodies were used as sandwich antibodies. Detection by the enzyme-linked immunosorbent assay required amplification of the virus. direct detection in clinical specimens (nasopharyngeal aspirates) was accomplished when MAbs recognizing two distinct antigenic sites of M1 were used in a time-resolved fluoroimmunoassay. Type A influenza viruses could be detected equally well in specimens obtained during epidemics of both H3N2 and H1N1 influenza viruses.
Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococcal group C disease in Canada
Authors: Ashton F E , Ryan J A , Borczyk A , Caugant D A , Mancino L , Huang D
Published: January 2020

Multilocus enzyme electrophoresis was used to characterize 378 isolates of Neisseria meningitidis serogroup C recovered during a period of an increase in group C meningococcal disease in Canada. Thirty-four enzyme electrophoretic types were found among the isolates, which were predominantly (96.0%) serotype 2a. One clone (ET 15), characterized by a rarely occurring allele for the enzyme fumarase, was responsible for a focal outbreak in Ontario followed by the spread of group C disease across the province. This clone, which occurred infrequently among strains isolated in 1986, accounted for over 65% of group C strains associated with meningococcal disease in Canada in 1990.
Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae
Authors: Shirai H , Nishibuchi M , Ramamurthy T , Bhattacharya S K , Pal S C , Takeda Y
Published: January 2020

We report a set of oligonucleotide primers and amplification conditions for the polymerase chain reaction to detect the ctx operon of Vibrio cholerae. The results of this assay on strains of V. cholerae and related organisms were identical with those obtained by the DNA colony hybridization test with the polynucleotide probe. The detection limit of this system was 1 pg of chromosomal DNA or broth culture containing three viable cells. The polymerase chain reaction method could directly detect the ctx operon sequences in rice-water stool samples from patients with cholera.
Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque
Authors: van Poperin N , Lopatin D E
Published: January 2020

A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development with BCIP-NBT substrate resulted in an easily discernible, permanent stain being deposited at the sample application site. Comparison with known concentrations of pure, cultured microorganisms applied to the same membranes permitted qualitative or semiquantitative plaque characterization by visual inspection. Analysis of the blots with a computer-linked flatbed scanner provided quantitative data on microbial content. The reproducibility of the results (standard error of the mean, less than 10%) obtained with slot immunoblotting greatly exceeded that of the results obtained with immunofluorescence analysis (standard error of the mean, greater than 57%). Because it is versatile, rapid, sensitive, reproducible, permanent, and relatively inexpensive, slot immunoblotting lends itself to use in large-scale investigations for the detection and quantitation of specific microbial species.
Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis
Authors: van Soolingen D , Hermans P W , de Haas P E , Soll D R , van Embden J D
Published: January 2020

In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.
Comparative evaluation of three selective media and a nonselective medium for the culture of Helicobacter pylori from gastric biopsies
Authors: Tee W , Fairley S , Smallwood R , Dwyer B
Published: January 2020

Plating on solid media is the standard technique used in most laboratories for the isolation of Helicobacter pylori from gastric biopsies. Recently, various selective media were developed for this purpose. We compared and evaluated three selective media, Skirrow's, Dent's CP, and modified Glupczynski's Brussels campylobacter charcoal media, and chocolate agar medium for the isolation of H. pylori. Gastric biopsies taken from a total of 203 patients were plated in parallel on all four media. An isolation rate of 51% (104 of 203) was obtained with a combination of all four media. Of the 104, 92 (88%) were positive with Dent's medium and with modified Glupczynski's medium. Skirrow's medium gave the highest isolation rate, 96% (100 of 104). However, growth of H. pylori was scant (only one to five colonies) when growth occurred on Skirrow's medium alone. Overall, modified Glupczynski's medium provided significantly heavier growth. Chocolate agar medium yielded a 76% (79 of 104) positivity rate. We recommend the use of a combination of two selective media for the maximum recovery of H. pylori from antral biopsies.
Characterization of six new capsular types (23 through 28) of Streptococcus suis
Authors: Gottschalk M , Higgins R , Jacques M , Beaudoin M , Henrichsen J
Published: January 2020

Six new capsular types of Streptococcus suis (types 23 to 28) are described. All reference strains were isolated from diseased pigs and were morphologically and biochemically similar to previously described capsular types 1 to 22. Clear and specific reactions were obtained for each of the new capsular types with three different typing techniques; no cross-reactions were detected among them or with other S. suis capsular types. Their capsular material presented similar ultrastructural characteristics, as shown by electron microscopy, and fimbriae similar to those described for other capsular types of S. suis were observed. When untypeable field isolates were tested with antisera raised against the six new capsular types, capsular type 23 appeared to be the most prevalent, representing more than 50% of all these isolates. Most isolates were recovered from cases of pneumonia, septicemia, and meningitis. Presumptive biochemical identification described for S. suis capsular types 1 to 22 may also be used for capsular types 23 to 28.
Evidence for long-term memory of the mucosal immune system: milk secretory immunoglobulin A against Shigella lipopolysaccharides
Authors: Hayani K C , Guerrero M L , Ruiz-Palacios G M , Gomez H F , Cleary T G
Published: January 2020

Although the common mucosal immune system has generally been considered to have only short-term memory, recent data suggest that long-term memory exists for Shigella virulence plasmid antigens. Because such antigens might cross-react with environmental antigens, we investigated milk for the persistence of antibodies to the specific Shigella lipopolysaccharide (LPS) antigens. Enzyme-linked immunosorbent assays to detect secretory immunoglobulin A (sIgA) against Shigella flexneri and Shigella sonnei LPS in milk samples were developed; 15 random milk samples tested on different days correlated from one day to the next (P = 0.0001). Of 18 Mexican mothers, 18 (100%) had one or more milk samples positive for anti-S. flexneri LPS, 14 (78%) had one or more milk samples positive for anti-S. sonnei LPS, and 14 (78%) had one or more milk samples positive for both. Of 27 Houston mothers, 16 (59%) had one or more milk samples positive for anti-S. flexneri LPS, 7 (26%) had one or more milk samples positive for anti-S. sonnei LPS, and 5 (19%) had one or more milk samples positive for both. Mexican mothers were significantly more likely than Houston mothers to have at least one sample with a positive titer for anti-S. flexneri LPS (P less than 0.02) and at least one sample with a positive titer for anti-S. sonnei LPS (P less than 0.002). Although the Houston women had a lower rate of titer positivity for both Shigella species, the rate was too high to be consistent with short-lived mucosal immunity. It is unlikely that 18 of the 27 Houston women had shigellosis during or just prior to lactation. The data suggest that there exists a long-term hormonally driven memory in the secretory immune system for Shigella spp.
Improved adhesive method for microscopic examination of fungi in culture
Authors: Rodriguez-Tudela J L , Aviles P
Published: January 2020

A new method for the examination of molds that involves the use of a device that dispenses a thin layer of a transparent adhesive material over the surface of a coverslip is described. The advantages of this method over previous methods used for the microscopic examination of molds are delineated.
Isolation, characterization, and serial propagation of a bovine group C rotavirus in a monkey kidney cell line (MA104)
Authors: Tsunemitsu H , Saif L J , Jiang B M , Shimizu M , Hiro M , Yamaguchi H , Ishiyama T , Hirai T
Published: January 2020

A virus (designated the Shintoku strain) which was morphologically indistinguishable from group A rotaviruses was detected in the feces of adult cows with diarrhea in Japan. The virus contained 11 segments of double-stranded RNA and had an electrophoretic migration pattern in polyacrylamide gels similar to that of other group C rotaviruses (4-3-2-2). Feces containing the bovine virus reacted with antiserum to porcine group C rotavirus (Cowden strain) but not group A or B rotaviruses in immunoelectron microscopy. The virus was adapted to serial propagation in roller tube cultures of a rhesus monkey kidney cell line (MA104) by using high concentrations of trypsin. Evidence for viral replication in MA104 cell cultures was demonstrated by immunoelectron microscopy and indirect immunofluorescence by using antiserum to porcine group C rotavirus and by electrophoretic analysis of extracted viral double-stranded RNA. A significant antibody response against the isolate was detected in convalescent-phase sera of cows which excreted the virus: no increased antibody response to bovine group A rotavirus was observed. To our knowledge, this is the first isolation of a group C rotavirus from cattle.
Diagnosis of acute pulmonary toxoplasmosis by visualization of invasive and intracellular tachyzoites in Giemsa-stained smears of bronchoalveolar lavage fluid
Authors: Bottone E J
Published: January 2020

Two patients with AIDS presented with pulmonary toxoplasmosis. Diagnosis was achieved by the visualization, in Giemsa-stained smears of bronchoalveolar lavage fluid, of tachyzoites of Toxoplasma gondii that were extracellular and in the process of invading or within pneumocytes. The intracellular localization of tachyzoites facilitated diagnosis by obviating potential confusion of extracellular tachyzoites with cellular debris or platelets.
Identification of Salmonellae with the 4-methylumbelliferyl caprilate fluorescence test
Authors: Olsson M , Syk A , Wollin R
Published: January 2020

We have tested 750 Salmonella strains and 130 strains of other species of the family Enterbacteriaceae with the 4-methylumbelliferyl caprilate reagent (MUCAP) test. The MUCAP test is a fluorescence test for rapid identification of Salmonella strains. The non-Salmonella strains were strains sent for identification as suspected Salmonella strains and thus have phenotypes similar to those of Salmonella strains. All 748 tested Salmonella strains of subgroups I, II, III, and IV were positive in the MUCAP test. Of the two tested rare Salmonella subgroup V strains, one was positive and the other was negative. In the selected material containing strains with phenotypes similar to those of Salmonella strains, only one Hafnia alvei strain of 130 Enterbacteriaceae bacteria tested was positive. The fluorescence of the H. alvei strain, the six tested Salmonella dublin strains, and the positive Salmonella subgroup V strain was weaker than that of the other salmonellae. The MUCAP assay is simple and is performed within 5 min. With an almost 100% sensitivity for Salmonella strains, apart from a single Salmonella subgroup V strain, we found the MUCAP test to be a convenient complement to traditional biochemical identification methods, especially for atypical and unusual Salmonella strains.
Nontoxigenic strains of Clostridium difficile lack the genes for both toxin A and toxin B
Authors: Fluit A C , Wolfhagen M J , Verdonk G P , Jansze M , Torensma R , Verhoef J
Published: January 2020

A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR). All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay. All nontoxigenic strains failed to produce toxin and were negative in the PCR assay. This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.
Genomic DNA fingerprinting by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus
Authors: Ichiyama S , Ohta M , Shimokata K , Kato N , Takeuchi J
Published: January 2020

In this study, we have compared genomic DNA fingerprintings among isolates of methicillin-resistant Staphylococcus aureus (MRSA) by using pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with SmaI were most suitable for the PFGE separation. SmaI cut genomic DNA into 15 to 20 fragments whose sizes ranged from about 30 to 1,500 kb. Thirty-one distinctive fragment patterns were identified in 111 infecting and colonizing MRSA isolates from six different hospitals in Japan. On the basis of the genomic typing by PFGE, we performed an epidemiological investigation of an outbreak of nosocomial MRSA infections among inpatients in Nagoya University Hospital. Ten types of chromosomal digestion were identified in the 20 strains isolated from 18 infected patients and 1 from colonized hospital personnel. According to the restriction patterns, we found that four types of these strains had caused epidemic infections among 13 patients in the outbreak. Two types (types 1 and 4) of the strains were involved in the death of five patients. The other infections were sporadic. The clarity and polymorphism of the chromosomal digestion patterns enabled us to discriminate between isolates which could not be differentiated by antibiogram or plasmid analysis. Classification of the genomic DNA fingerprinting patterns by PFGE is therefore proposed as a useful method for investigating the source, transmission, and spread of nosocomial MRSA infections.
Comparison of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus
Authors: Espy M J , Wold A D , Jespersen D J , Jones M F , Smith T F
Published: January 2020

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.
Quantitative microculture system with standardized inocula for strain typing, susceptibility testing, and other physiologic measurements with Candida albicans and other yeasts
Authors: Odds F C
Published: January 2020

A method for physiologic testing of Candida albicans and other yeasts is described in which inocula with reproducible cell concentrations are prepared from cultures in a glucose-limited medium and growth turbidities of test cultures in microplates are expressed quantitatively as a percentage of control growth. The method gave good within-plate and run-to-run reproducibilities and was applicable both to strain phenotype discrimination in C. albicans and to susceptibility tests of yeasts with azole antifungal agents.
Detection of variable DNA repeats in diverse eukaryotic microorganisms by a single set of polymerase chain reaction primers
Authors: Riley D E , Samadpour M , Krieger J N
Published: January 2020

We cloned and sequenced a variable DNA repeat from Trichomonas vaginalis, a flagellated protozoan parasite. Targeting of this repeat in the polymerase chain reaction resulted in complex and intense product patterns for a wide variety of eukaryotic microorganisms, including the pathogenic protozoan parasites T. vaginalis, Giardia lamblia, Leishmania donovani, three species of Trypanosoma, and four species of Acanthamoeba; the nonpathogenic protozoans, Paramecium tetraurelia and Tetrahymena thermophilia; and a yeast, Saccharomyces cerevisiae. Each microorganism exhibited a distinctive pattern of repeats. For example, a characteristic pattern was exhibited by six clinical T. vaginalis isolates. Eight G. lamblia isolates exhibited either one of two characteristic pattern types. There was no reaction with human DNA or DNA from the prokaryotes Ureaplasma urealyticum and Mycoplasma hominis. This approach may facilitate detection of a wide variety of eukaryotic microorganisms by use of a single primer set and holds promise for the development of typing schemes for both T. vaginalis and G. lamblia.
Babesia microti, human babesiosis, and Borrelia burgdorferi in Connecticut
Authors: Anderson J F , Mintz E D , Gadbaw J J , Magnarelli L A
Published: January 2020

Babesia microti was isolated from a white-footed mouse (Peromyscus leucopus) that was captured in southeastern Connecticut in 1988, when the first human case of babesiosis acquired in Connecticut was recognized. To date, 13 cases of babesiosis have been reported in Connecticut, the largest number of human cases reported on the mainland United States. Two of nine patients quiried remembered a prior tick bite. Since Babesia parasites are known to be vectored only by ticks, we surmise that 12 of these infections were acquired via tick bites; 1 was obtained by blood transfusion (the patient was 46 years of age) from an endemically infected donor. The ages of the patients with tick-acquired babesiosis ranged from 61 to 95 years. Two patients died with active infections, and one patient died from chronic obstructive pulmonary disease soon after treatment with clindamycin and quinine. Indirect fluorescent-antibody titers of blood samples drawn at the time of hospitalization for 11 patients and at the time of active infection for 1 asymptomatic person ranged from 1:1,024 to 1:4,096. Five of eight patients with babesiosis also had significant immunoglobulin G or immunoglobulin M titers (1:640 to 1:5,120) to Borrelia burgdorferi. B. microti was isolated in Syrian hamsters inoculated with blood from 7 of 12 patients tested and was also isolated from mice captured in six towns. The peridomestic nature of the disease was demonstrated by isolating the parasite from white-footed mice captured in or near the yards of eight different patients. Of 59 mice tested, 27 were positive and 25 were coinfected with B. burgdorferi. The isolation of B. microti from a white-footed mouse captured in north-central Connecticut (West Hartford), away from the focus of human infections in southeastern Connecticut, suggests that this pathogen may spread into other areas where Ixodes dammini, the tick vector, becomes established.