bluuebunny/crossref_metadata_2025 huggingface dataset:
Current practices in mycobacteriology: results of a survey of state public health laboratories
Authors: Huebner R E , Good R C , Tokars J I
Published: January 2020

Fifty-six state and territorial public health laboratories were surveyed to determine whether currently available rapid methods for the identification and drug susceptibility testing of Mycobacterium tuberculosis were being performed. Forty (71%) laboratories use fluorochrome rather than conventional basic fuchsin stains for screening clinical specimens for acid-fast bacilli. Of the 55 laboratories that routinely culture for mycobacteria, 16 (29%) use the more rapid radiometric methods. Species identification of isolates is done by biochemical tests in 13 (23%) laboratories; 40 (72%) use nucleic acid probes, high-performance liquid chromatography, or the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test (rapid tests); 3 laboratories do not perform species identification. Drug susceptibility testing is performed with solid media by 36 of 45 (80%) laboratories, while the more rapid radiometric methods are used by 9 (20%) laboratories. Compared with the laboratories that use conventional methods, laboratories that use rapid methods report results more quickly: for species identification, 43 days (conventional) versus 22 days (rapid); for drug susceptibility testing, 44 days (conventional) versus 31 days (rapid) from specimen processing. Rapid technologies for microscopy and species identification are being used by many, but not all, state and territorial public health laboratories; however, most laboratories do not use the more rapid radiometric methods for routine culture or drug susceptibility testing of mycobacteria. Implementation of such rapid technologies can shorten turnaround times for the laboratory diagnosis of tuberculosis and recognition of drug resistance.
Detection of Helicobacter pylori in dental plaque by reverse transcription-polymerase chain reaction
Authors: Nguyen A M , Engstrand L , Genta R M , Graham D Y , el-Zaatari F A
Published: January 2020

To investigate whether the oral cavity is a potential reservoir and possible sanctuary for Helicobacter pylori, supragingival and subgingival plaques were analyzed by a Helicobacter genus-specific reverse transcriptase-polymerase chain reaction based on the sequence data of H. pylori 16S rRNA. The amplified 500-bp DNA fragment was identified by ethidium bromide staining after agarose gel electrophoresis and by Southern hybridization. Twenty-five dyspeptic patients were studied. Histologic examination of gastric biopsy specimens revealed that 18 had H. pylori gastritis and 7 did not. For seven of the 18 (38.8%) patients with proven H. pylori gastritis, H. pylori was also identified in their dental plaque. None of the patients without H. pylori gastritis had H. pylori in their dental plaque. The detection of H. pylori in dental plaque suggests that this H. pylori colonization is not restricted to the gastric mucosa and that this ecological niche may serve as a possible sanctuary which may be responsible for reinoculation of the stomach after topical anti-H. pylori therapies such as bismuth.
Marked phenotypic variability in Pseudomonas cepacia isolated from a patient with cystic fibrosis
Authors: Larsen G Y , Stull T L , Burns J L
Published: January 2020

Characterization of the epidemiology of Pseudomonas cepacia colonization in cystic fibrosis is difficult because of the phenotypic variability of isolates. A single sputum culture may yield colonies which differ in morphology, antibiotic susceptibility, and pigment production. We examined serial P. cepacia isolates from a cystic fibrosis patient which the clinical laboratory identified as separate strains; these were selected on the basis of isolation date and culture site. An attempt was made to sample at multiple time points and, at a single time point, from three different culture sites. Ribotype analysis, using both the standard Southern blot technique and a recently reported method which uses the polymerase chain reaction, was used to distinguish unique P. cepacia strains. Characterization included comparison of antibiotic susceptibility, plasmid content, and outer membrane protein (OMP) patterns. rRNA analysis demonstrated that all isolates had the same ribotype, consistent with their being derivatives of the same strain. Antibiotic susceptibility testing revealed variability among both same-date and same-site isolates. Screening for plasmid DNA identified three groups of isolates; both same-date and same-site isolates demonstrated variability. OMP profiles were similar, but at least six distinct patterns were identified. For the six same-date isolates, five different OMP patterns were identified. For the 10 same-site isolates from different dates, five of the six OMP patterns were represented. We have demonstrated marked phenotypic variability in 14 strains of P. cepacia isolated from different sites and at different times from a single colonized patient. Ribotyping identified all the isolates as derivatives of a single strain; thus, the diversity of phenotypes appears to be the result of differential gene expression.
Role of group C beta-hemolytic streptococci in pharyngitis: epidemiologic study of clinical features associated with isolation of group C streptococci
Authors: Turner J C , Fox A , Fox K , Addy C , Garrison C Z , Herron B , Brunson C , Betcher G
Published: January 2020

All Lancefield group C beta-hemolytic streptococci isolated over 12 months from college students with clinical pharyngitis and age-matched healthy controls were identified. Clinical features of upper respiratory tract infection and pyogenic pharyngitis as well as colony counts were tabulated for each patient according to throat culture results. Of 1,480 patients, Lancefield group C Streptococcus equisimilis was isolated from 45 (3%) patients and Streptococcus anginosus ("Streptococcus milleri") was isolated from 164 (11.1%) patients. Patients from whom S. equisimilis was isolated had clinical features more suggestive of pyogenic infection than did patients from whom S. anginosus was isolated. Colony counts on primary throat culture plates from patients from whom S. equisimilis and Streptococcus pyogenes were isolated were higher than those from patients from whom S. anginosus was isolated. This study presents epidemiologic evidence supporting a role for S. equisimilis in causing pharyngeal infection and for S. anginosus as representing part of the normal oropharyngeal flora.
Improved recovery of mycobacteria from respiratory secretions of patients with cystic fibrosis
Authors: Whittier S , Hopfer R L , Knowles M R , Gilligan P H
Published: January 2020

Pulmonary colonization and infection of patients with cystic fibrosis by Mycobacterium spp. has recently been recognized as a potentially important clinical problem. However, frequent contamination of mycobacterial cultures by pseudomonads has hampered efforts to define the extent of this problem. This study was done to evaluate current techniques and to establish a more efficient method of recovering mycobacteria from respiratory secretions of patients with cystic fibrosis. Decontamination of respiratory specimens (n = 121) with 0.25% N-acetyl-L-cysteine and 1% sodium hydroxide (NALC-NaOH) was associated with a high rate of pseudomonas overgrowth for both Lowenstein-Jensen slants (74%) and BacTec vials supplemented with PANTA (polymyxin B [50 U/ml], amphotericin B [5 micrograms/ml], nalidixic acid [20 micrograms/ml], trimethoprim [5 micrograms/ml], azlocillin [10 micrograms/ml]) (36%). This overgrowth limited recovery of mycobacteria to only 64% (9 of 14) of specimens positive by smear for acid-fast bacilli (AFB). Decontamination of specimens (n = 441) with NALC-NaOH, followed by 5% oxalic acid treatment, resulted in contamination of only 5% of Lowenstein-Jensen slants and 3% of BacTec vials. AFB were recovered from all 90 AFB smear-positive specimens following the use of this decontamination technique. We recommend that respiratory secretions be decontaminated with NALC-NaOH and oxalic acid to decrease the incidence of Pseudomonas aeruginosa overgrowth.
Rapid, polymerase chain reaction-based identification assays for Candida species
Authors: Niesters H G , Goessens W H , Meis J F , Quint W G
Published: January 2020

Polymerase chain reaction (PCR) amplification of specific regions in the genomes of a variety of lower eukaryotes permits rapid identification of these microorganisms. First, on the basis of the presence of both constant and variable regions in the small subunit (ssu) rRNA, a nested PCR for direct identification of various Candida species can be designed. Amplification of the entire ssu rRNA gene and subsequent reamplification of variable sequences within the V4 domains of these PCR products were combined with direct sequencing. Restriction enzyme maps were made, and species-specific oligonucleotides for hybridization analysis were selected. Unequivocal discrimination of four of the major human pathogenic yeasts (Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei) is possible if a combination of these techniques is used. Second, by using oligonucleotides aimed at repeated sequences which occur at dispersed positions in the genomes of all eukaryotes, species-specific DNA fingerprints could be generated. This interrepeat PCR using genomic DNA as template proved to be an effective tool in Candida species typing. Both techniques described here can be extrapolated to the high-speed diagnostics of numerous other prokaryotic and eukaryotic pathogens.
Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens
Authors: Ishida C , Matsumoto K , Fukada K , Matsushita K , Shiraki H , Maeda Y
Published: January 2020

We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein.
Increased specificity of antibody detection in surgical patients with invasive candidiasis with cytoplasmic antigens depleted of mannan residues
Authors: van Deventer A J , van Vliet H J , Voogd L , Hop W C , Goessens W H
Published: January 2020

In this study, it was shown that the diagnostic accuracy of antibody detection by a counterimmunoelectrophoresis technique could be improved by using cytoplasmic antigens depleted of mannan residues. The specificity of the counterimmunoelectrophoresis increased from 28.6 to 78.6% when cytoplasmic antigens depleted of mannan were used, while the sensitivity slightly decreased from 80 to 70%.
Rejection criteria for endotracheal aspirates from adults
Authors: Morris A J , Tanner D C , Reller L B
Published: January 2020

Although criteria have been established to assess the quality of sputum specimens, no criteria for assessing the quality of endotracheal suction aspirates (ETSA) exist. Therefore, we compared the Gram stain (GS) and culture results for 504 consecutive ETSA specimens. Results recorded for GS included the numbers of squamous epithelial cells (SEC) and polymorphonuclear leukocytes (PML) per low-power field (LPF) (magnification, x100) as well as the quantities and types of organisms per high-power field (HPF) (magnification, x1,000). Culture results were quantitated by organism. Only 15% of ETSA specimens tested by GS contained > 10 SEC per LPF, and 21, 20, and 59% had < or = 10, 11 to 24, and > or = 25 PML per LPF, respectively. For 40% of ETSA specimens, no organisms were visible by GS. Of these specimens, 40% were sterile, 48% grew normal oropharyngeal flora (NF) only, 5% grew 1+ NF (i.e., > 10 colonies in the first quadrant) and 1+ gram-negative rods (GNR), and 7% grew < or = 1+ GNR either alone or in mixed culture. The mean numbers of organisms recovered from ETSA with < or = 10 SEC per LPF and > 10 SEC per LPF were 2.35 and 4.05, respectively. We therefore recommend that ETSA specimens that show no organisms by GS be rejected, in addition to those with > 10 SEC per LPF. Application of these rejection criteria enabled us to reject 847 (41%) of 2,068 ETSA specimens over a 6-month period. This represents a saving of approximately $66,000/year in unnecessary laboratory charges to patients.
Identification of Leuconostoc spp. by analysis of soluble whole-cell protein patterns
Authors: Elliott J A , Facklam R R
Published: January 2020

Leuconostoc spp. share several physiologic characteristics, which sometimes makes it difficult to identify these organisms to the species level. We developed a system, based on the patterns of soluble whole-cell proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that was able to discriminate between the six Leuconostoc spp. that are capable of growth at 37 degrees C. Nine unidentified Leuconostoc-like bacterial isolates that were included in the study did not fit any of the protein profiles of the type strains and may represent new Leuconostoc spp.
Polymerase chain reaction for detection of measles virus in clinical samples
Authors: Shimizu H , McCarthy C A , Smaron M F , Burns J C
Published: January 2020

A rapid and sensitive one-step reverse transcription polymerase chain reaction assay was developed to detect measles virus (MV) in nasal aspirates from patients with suspected MV infection. Oligonucleotide primers and probe were targeted to highly conserved regions of the matrix gene. Assay conditions were optimized to allow detection of as little as 1 PFU of an MV stock whose titer was known. Extraction of RNA from 38 nasal aspirates and then reverse transcription and MV matrix gene amplification yielded a polymerase chain reaction product of the predicted size in 14 of 14 MV culture-positive patients. Matrix gene amplification provides a rapid, sensitive, and specific supplementary assay to the currently available modalities for MV detection.
Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay
Authors: He Y , Coutlée F , Saint-Antoine P , Olivier C , Voyer H , Kessous-Elbaz A
Published: January 2020

An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.
Peptide antisera targeted to a conserved sequence in poliovirus capsid VP1 cross-react widely with members of the genus Enterovirus
Authors: Hovi T , Roivainen M
Published: January 2020

Rabbits were immunized with synthetic peptides derived from an immunodominant region of the VP1 capsid protein of enteroviruses. This region shows a high degree of homology among all sequenced members of the genus. Peptide-induced antisera were used for immunoperoxidase staining of cell cultures infected with 41 different serotypes of enterovirus. Specific cytoplasmic staining was readily seen in all but two cases. Echovirus type 22 was previously known to differ genetically from the rest of the enteroviruses, and hence, a negative result was expected. Surprisingly, one of the tested serum samples reacted with echovirus 22-infected cells. Coxsackievirus A7-infected cells could be reliably stained with only one of the tested serum samples. For the remaining 39 serotypes, scattered infected cells resulting from 1 to 2 days of incubation with diluted inocula were easily scored as positive before the cytopathic effect became visible. The same antibodies were also used in a sandwich-type enzyme immunoassay to demonstrate poliovirus antigens in cell extracts as early as 3 h after a high-multiplicity infection. These antibodies are candidates for enterovirus group reagents, being potentially useful in both the laboratory diagnosis of enterovirus infections and research on enterovirus-host interactions.
Differentiation of Salmonella phase 1 flagellar antigen types by restriction of the amplified fliC gene
Authors: Kilger G , Grimont P A
Published: January 2020

The large antigenic diversity (over 2,300 serotypes) expressed by Salmonella strains can probably be observed at the genetic level. The phase 1 flagellin gene fliC was amplified, and the amplified fragment was cleaved with a mixture of both endonucleases TaqI and ScaI. The restriction patterns observed allowed differentiation of flagellar types b, i, d, j, l,v, and z10. Flagellar group g (g,m, g,p, or g,m,s) could be differentiated from the other flagellar types. Flagellar types r and e,h could not be separated, although they could be distinguished from the other flagellar types studied. Practical applications of flagellar gene restriction are the distinction between serotype Gallinarum-Pullorum, which carries a cryptic gene for flagellar type g,m, and nonmotile Vi-negative variants of serotype Typhi, and the tentative assignation of nonmotile variants of Salmonella serotypes to a flagellar type.
Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections
Authors: Quentin R , Martin C , Musser J M , Pasquier-Picard N , Goudeau A
Published: January 2020

In recent years, reports originating from several areas of the world have identified biotype IV strains of Haemophilus influenzae as a cause of serious urogenital, neonatal, and mother-infant infections. Preliminary analysis of a sample of biotype IV isolates found evidence for a cryptic genospecies of Haemophilus (R. Quentin, A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. J. Gen. Microbiol. 136:1203-1209, 1990). Eighteen biotype IV strains assigned to the cryptic genospecies were further characterized by their rDNA restriction fragment length polymorphism patterns and genomic DNA-DNA hybridization. Isolates of the cryptic genospecies have distinctive rDNA restriction fragment length polymorphisms that differ from those of Haemophilus haemolyticus and H. influenzae. Genomic hybridization studies show that these organisms are allied with H. influenzae and H. haemolyticus and suggest a distant trifurcation of H. influenzae, H. haemolyticus, and the cryptic genospecies.
Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses
Authors: Fritsche T R , Gautom R K , Seyedirashti S , Bergeron D L , Lindquist T D
Published: January 2020

Free-living and parasitic protozoa are known to harbor a variety of endosymbiotic bacteria, although the roles such endosymbionts play in host survival, infectivity, and invasiveness are unclear. We have identified the presence of intracellular bacteria in 14 of 57 (24%) axenically grown Acanthamoeba isolates examined. These organisms are gram negative and non-acid fast, and they cannot be cultured by routine methodologies, although electron microscopy reveals evidence for multiplication within the amoebic cytoplasm. Examination for Legionella spp. with culture and nucleic acid probes has proven unsuccessful. We conclude that these bacteria are endosymbionts which have an obligate need to multiply within their amoebic hosts. Rod-shaped bacteria were identified in 5 of 23 clinical Acanthamoeba isolates (3 of 19 corneal isolates and 2 of 4 contact lens isolates), 4 of 25 environmental Acanthamoeba isolates, and 2 of 9 American Type Culture Collection Acanthamoeba isolates (ATCC 30868 and ATCC 30871) previously unrecognized as having endosymbionts. Coccus-shaped bacteria were present in one clinical (corneal) isolate and two environmental isolates. There was no statistical difference (P > 0.8) between the numbers of endosymbiont strains originating from clinical (26% positive) and environmental (24% positive) amoebic isolates, suggesting that the presence alone of these bacteria does not enhance amoebic infectivity. Rods and cocci were found in both clinical and environmental isolates from different geographical areas (Seattle, Wash., and Portland, Oreg.), demonstrating their widespread occurrence in nature. Our findings suggest that endosymbiosis occurs commonly among members of the family Acanthamoebidae and that the endosymbionts comprise a diverse taxonomic assemblage. The role such endosymbionts may play in pathogenesis remains unknown, although a variety of exogenous bacteria have been implicated in the development of amoebic keratitis, warranting further evaluation.
Isolation of gram-positive rods that resemble but are clearly distinct from Actinomyces pyogenes from mixed wound infections
Authors: Wüst J , Lucchini G M , Lüthy-Hottenstein J , Brun F , Altwegg M
Published: January 2020

Beginning in 1990, gram-positive rods resembling Actinomyces pyogenes were found with increasing frequency in mixed cultures from various infectious processes, most of them from patients with otitis, empyema, pilonidal cysts, perianal abscesses, and decubitus ulcers. Ribotyping and hybridization showed that these gram-positive rods could be divided into five groups not related to known Actinomyces species. Biochemical markers for reliable differentiation into these groups, however, could not be found. Therefore, naming new species is not warranted unless parameters are discovered that allow identification without DNA hybridization. These gram-positive rods have been isolated only in mixed cultures with anaerobes, Staphylococcus aureus, Streptococcus "milleri," enterococci, and gram-negative rods. Their exact role in these possibly synergistic infections needs further investigation.
Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction
Authors: Hennessy K J , Iandolo J J , Fenwick B W
Published: January 2020

Rapid and accurate determination of the Actinobacillus pleuropneumoniae serotype involved in a disease outbreak is important both in limiting the severity of an outbreak and for tracing the source of the infecting organism. This study describes the use of arbitrarily primed polymerase chain reaction (AP-PCR) as a rapid, precise, and genetically based procedure to identify A. pleuropneumoniae. AP-PCR amplification of bacterial genomic DNA results in specific DNA profiles, which can be used to differentiate currently recognized serotypes. This technique is especially useful for identifying previously nontypeable and serologically cross-reactive A. pleuropneumoniae field isolates. Consecutive passages of isolates on different media, freezing, and subsequent infection of pigs did not alter the AP-PCR genomic profile. We propose the use of M13 and T3-T7 oligodeoxynucleotide primers for diagnostic and epidemiological identification of A. pleuropneumoniae by AP-PCR techniques.
Collaborative evaluation of the Radiometer Sensititre AP80 for identification of gram-negative bacilli
Authors: Staneck J L , Weckbach L S , Tilton R C , Zabransky R J , Bayola-Mueller L , O'Hara C M , Miller J M
Published: January 2020

A multicenter trial of the Sensititre AP80 panel read on the Sensititre AutoReader (Radiometer America, Westlake, Ohio) for the automated identification of gram-negative bacilli was conducted with 1,023 clinical isolates (879 members of the family Enterobacteriaceae plus 144 nonenteric organisms). Assignment of taxa was based on the computer-assisted interpretation of the results of a series of reactions with fluorogenic enzyme substrates after 5 h of incubation, with an incubation interval of approximately 18 h used when indicated. Accuracy was determined initially by comparison with the results obtained with the API 20E or Rapid NFT system (Analytab Products, Plainview, N.Y.). Isolates showing discrepancies were identified by using conventional biochemical profiles. Identifications were available after 5 h of incubation for 918 isolates (90%). Agreements with reference results for members of the family Enterobacteriaceae were 95.3 and 92.5% at the genus and species levels, respectively, and for the nonmembers of the family Enterobacteriaceae, the agreements with reference results were 95.1 and 84.7%, respectively. The Sensititre AP80 panel was found to be simple and convenient to use, allowed for the testing of three isolates per panel, required minimal supplementary testing for completion of identification, performed in a reproducible fashion, and demonstrated an accuracy of same-day identification comparable to that reported for other automated systems. The AP80 panel appears well suited for routine use in the clinical microbiology laboratory as an automated means of identifying both members of the family Enterobacteriaceae and nonenteric gram-negative bacilli.
Expression of hemolytic activity by Plesiomonas shigelloides
Authors: Janda J M , Abbott S L
Published: January 2020

More than 90% of the Plesiomonas shigelloides strains that we tested produced a beta-hemolysin, as judged by the results of agar overlay and contact-dependent hemolysis assays. The hemolysin was cell associated, was active against the erythrocytes of various animal species, and was synthesized at both 25 and 35 degrees C. Activity was lost after thermal or proteolytic treatments or after preincubation in the presence of gentamicin; hemolytic activity did not appear to correlate with the previously established 50% lethal doses for seven of these strains. The hemolysin may play a role in iron acquisition in vivo via the lysis of erythrocytes, liberating hemoglobin, or, alternatively, may be involved in gastrointestinal disease.
Comparative study of procedures for isolation and cultivation of Legionella pneumophila from tap water in hospitals
Authors: Reinthaler F F , Sattler J , Schaffler-Dullnig K , Weinmayr B , Marth E
Published: January 2020

For the isolation and cultivation of Legionella pneumophila from tap water in hospitals, we compared different media and selection techniques. A second part of the study compared the L. pneumophila yields from different water samples at identical sites. A total of 210 water samples (500 ml each) were collected from two selected sites in each of 21 hospitals. Warm water samples were collected after flow times of 0, 5, 10, and 15 min; in addition, one cold water sample was collected. Filtration was used to concentrate all samples. Following filtration, 0.1 and 1 ml each of untreated samples, heat-treated samples (3 min, 59 degrees C), and acid-treated samples (pH 2.2, 15 min) were spread onto the selective media MWY (SR 118; Oxoid) and BMPA alpha (SR 111; Oxoid), and samples from 12 hospitals were also spread onto GVPC medium (SR 152; Oxoid). A total of 72 (34%) of the 210 samples from 12 hospitals were positive. With respect to the positive Legionella cultures, there was no significant difference between the selective media MWY, BMPA alpha, and GVPC. With the BMPA alpha supplement, more samples were positive following heat treatment (P < 0.05) or acid treatment (P < 0.05) than without any further treatment. For the maximum yield of Legionella colonies with minimum additional microbial flora, acid treatment was the most effective, and by all methods, the GVPC supplement was the most selective. For routine water tests in hospitals for differentiating between systemic and local contamination, acid treatment of the concentrated samples, the use of different selective media, and the correct selection of sampling sites are recommended.
Analysis of respiratory syncytial virus genetic variability with amplified cDNAs
Authors: Sullender W M , Sun L , Anderson L J
Published: January 2020

Antigenic and genetic heterogeneities exist within the two major antigenic groups of respiratory syncytial (RS) virus. We developed a polymerase chain reaction (PCR)-based assay that not only differentiates the two RS virus groups but allows distinctions within groups on the basis of changes in the nucleotide sequences, as revealed by restriction fragment analysis. In this assay, viral RNA served as a template for cDNA synthesis with extension from a synthetic oligonucleotide primer complementary to bases 164 to 186 in the F protein mRNA. For PCR amplification, two group-specific 5' primers were added. The two primers corresponded to the G protein mRNA sequence of group B (bases 10 to 30) or group A (bases 247 to 267) RS virus. Agarose gel electrophoresis readily discriminated the 1.1-kb group B and the 0.9-kb group A virus amplification products. All 47 viruses tested were assigned to the same group by both PCR and monoclonal antibody reaction pattern analysis. Restriction fragment analysis of the amplified DNAs revealed 12 restriction patterns for group A viruses and 7 restriction patterns for group B viruses, while the monoclonal antibody reaction patterns revealed seven patterns for group A viruses and 3 patterns for group B viruses. Most viruses with the same monoclonal antibody reaction patterns had different restriction patterns, and some viruses with the same restriction patterns had different monoclonal antibody reaction patterns. Thus, the results of the PCR assay concurred with the monoclonal antibody reaction pattern analysis for group classification of RS viruses, while the restriction fragment analysis identified greater diversity within groups than was seen with the monoclonal antibody analysis.
Fluorometric quantitation of broth-cultured mycoplasmas by using alkaline ethidium bromide
Authors: Schaeffer W I , Melamede R
Published: January 2020

We developed a fluorometric system which does for broth-grown mycoplasmas what turbidimetric analysis does for broth-grown bacteria. It allows one to monitor the growth of broth-grown mycoplasmas at any interval desired. The entire procedure is quick, taking not more than 20 min. The fluorometric readings correlate with colonial growth on agar, making it possible, for the first time, to take readings which closely estimate the CFU present in the culture at a given moment in time. We show that this system can be used to assess the effectiveness of an antimycoplasmal antibiotic and to optimize medium components and that fluorometer readings taken during the logarithmic phase of growth correlate with the DNA content of the viable cells. Use of this methodology will permit investigators to know absolutely the phase of the growth cycle of the culture concomitant with the growth of the culture itself, and since fluorometer readings of culture aliquots can be converted to DNA equivalents, the standardization of mycoplasmal cultures within and between laboratories will be a possibility.
Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene
Authors: Yoh M , Miyagi K , Matsumoto Y , Hayashi K , Takarada Y , Yamamoto K , Honda T
Published: January 2020

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.
Species distribution of coagulase-negative staphylococcal isolates at a community hospital and implications for selection of staphylococcal identification procedures
Authors: Kleeman K T , Bannerman T L , Kloos W E
Published: January 2020

A total of 499 coagulase-negative staphylococci (CoNS) were isolated from a variety of clinical specimens at a community hospital. Ten different species and many strains of CoNS were identified. Staphylococcus epidermidis was the most common isolate. The species distribution suggests that S. saprophyticus and, to a lesser extent, S. haemolyticus may be important in urinary tract infections. S. lugdunensis may be a significant isolate from wound infections. Frequently, mixed cultures were found with either multiple species or multiple strains of the same species of CoNS. These mixed cultures could not be detected by colony morphology upon initial overnight incubation of the cultures but could be distinguished following colony development for several days. In addition, sequential positive cultures from an individual patient often yielded different species or different strains of the same species which again could not be detected upon initial observations of colony morphology. Procedures for the identification of the CoNS need to be improved, and microbiology laboratories should consider the use of more definitive identification procedures for the CoNS.
Evaluation of the Vitek Systems Gram-Positive Identification card for species identification of coagulase-negative staphylococci
Authors: Bannerman T L , Kleeman K T , Kloos W E
Published: January 2020

Vitek Systems' Gram-Positive Identification test (GPI) card was evaluated for the ability to identify 12 coagulase-negative Staphylococcus species and subspecies. The bionumber generated from the GPI card was examined for its potential use in epidemiological studies. Results indicated that the GPI card had a high degree of correlation with the conventional methods of identification. The species identified with the greatest accuracy were Staphylococcus epidermidis (92%), S. haemolyticus (95%), S. capitis subsp. capitis (88%), and S. saprophyticus (100%). S. hominis (63%) was identified with the least accuracy. The bionumber was found to have limited epidemiological value because of the frequent occurrence of a few major bionumbers.
Comparative assessment of the leprosy antibody absorption test, Mycobacterium leprae extract enzyme-linked immunosorbent assay, and gelatin particle agglutination test for serodiagnosis of lepromatous leprosy
Authors: Escobar-Gutiérrez A , Amezcua M E , Pastén S , Pallares F , Cázares J V , Pulido R M , Flores O , Castro E , Rodríguez O
Published: January 2020

A comparative assessment of three serological methods for leprosy diagnosis (the fluorescent leprosy antibody absorption [FLA-ABS] test, the Mycobacterium leprae soluble-extract enzyme-linked immunosorbent assay [ELISA], and the M. leprae particle agglutination [MLPA] test) was carried out. The objective was to identify their performance in clinical and epidemiological diagnosis of leprosy. The study group included 45 lepromatous leprosy patients under treatment. Specificity was > 95% for all three assays, and sensitivity was 95, 58, and 74% for the FLA-ABS test, the MLPA test, and the ELISA, respectively. The only cross-reactivity for M. tuberculosis-infected patients was with the soluble-extract ELISA. Although the FLA-ABS test displayed the highest specificity and sensitivity values, it can only be used in well-developed laboratories, and the patient's clinical and epidemiological background must be considered when results are interpreted because the test remains positive after therapeutic success and could be positive for some household contacts. The MLPA test is easier to perform and interpret, and it is adequate for small laboratories and epidemiological studies intended to detect active untreated or irregularly treated leprosy cases. Therefore, the FLA-ABS and MLPA tests are complementary, and both should be used for serodiagnosis of leprosy.
Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples
Authors: Severini G M , Mestroni L , Falaschi A , Camerini F , Giacca M
Published: January 2020

A method based on nested polymerase chain reaction was developed for the detection of enteroviral genomes in biological samples. By taking advantage of the conserved 5' noncoding region of the enteroviral RNA, two sets of primers were utilized, enabling the detection either of a broad range of enteroviruses or of group B coxsackieviruses only. The sensitivity of the method is close to the detection of single molecules of viral RNA in as much as 1 mg of tissue sample. A preliminary study showed the usefulness of this technique for the analysis of endomyocardial biopsy samples from patients with idiopathic dilated cardiomyopathy and myocarditis.
Development of a polymerase chain reaction test for specific identification of the urinary tract pathogen Aerococcus urinae
Authors: Aguirre M , Collins M D
Published: January 2020

A polymerase chain reaction test was developed for identification of the gram-positive urinary tract pathogen Aerococcus urinae. Oligonucleotide primers were based on highly specific sequences within the small-subunit rRNA gene. A confirmatory test based on hybridization of the amplified products to a highly specific internal probe was also developed.
Anticandidal activity and interleukin-1 beta and interleukin-6 production by polymorphonuclear leukocytes are preserved in subjects with AIDS
Authors: Cassone A , Palma C , Djeu J Y , Aiuti F , Quinti I
Published: January 2020

Polymorphonuclear granulocytes (PMN; or neutrophils) from uninfected or human immunodeficiency virus-infected subjects were tested for their ability to inhibit growth of Candida albicans and produce interleukin-1 beta (IL-1 beta) and IL-6 in vitro. It was seen that PMN from AIDS (Centers for Disease Control stage IV) patients expressed equal if not greater anticandidal activity compared with the activity expressed by neutrophils from all other subjects examined. On exposure to granulocyte macrophage-colony-stimulating factor or to a mannoprotein constituent (MP-F2) from C. albicans itself, PMN from AIDS patients showed enhanced antifungal activity and production of remarkable quantities of IL-1 beta and IL-6. These findings suggest that the functional abilities of PMN to inhibit Candida growth and secrete relevant proinflammatory and immunomodulatory cytokines are intrinsically preserved in AIDS patients.
Development of an enzyme-linked immunosorbent assay method for typing and quantitation of Klebsiella pneumoniae lipopolysaccharide: application to serotype O1
Authors: Albertí S , Hernández-Allés S , Gil J , Reina J , Martínez-Beltrán J , Camprubí S , Tomás J M , Benedí V J
Published: January 2020

We describe a method for the typing and quantitation of Klebsiella pneumoniae serotype O1 lipopolysaccharide (LPS) based on inhibition in an enzyme-linked immunosorbent assay of a reaction of known O1 LPS antigen and anti-O1 antibody by unknown LPS extracts. Serotype O1 was found in 32% of the 124 K. pneumoniae clinical isolates tested, showing that this serotype is frequent among the eight O serotypes which have been described previously.
New method to assess dilution of secretions for immunological and microbiological assays
Authors: Virolainen A , Mäkelä M J , Esko E , Jero J , Alfthan G , Sundvall J , Leinonen M
Published: January 2020

Accurate quantitation of pathogens and antibody concentrations in secretions has been difficult because of unpredictable dilution of secretion with th diluent at the time of sample collection. We added an inert substance, lithium chloride (LiCl), to the sample diluent and measured its concentration with an atomic absorption spectrometer before and after the specimen was added. LiCl, at a concentration of 2 mmol of Li per liter, has no negative effect on the survival of common respiratory pathogens or on the results of immunoassays. The method is applicable to any sample collecting in which dilution of the specimen is necessary.
Antigenic and genetic characterization of Borrelia species isolated from Ixodes persulcatus in Hokkaido, Japan
Authors: Fukunaga M , Sohnaka M , Takahashi Y , Nakao M , Miyamoto K
Published: January 2020

Ten characteristic strains of spirochetes (HT2, HT7, HT10, HT15, HT17, HT19, HT20, HT22, HT32, and HT59) isolated from Ixodes persulcatus adult ticks in Hokkaido, Japan, were selected and analyzed by polyacrylamide gel electrophoresis, with monoclonal and polyclonal antibodies, and by pulsed-field gel electrophoresis. The protein profiles of the borrelial isolates were variable and markedly different from that of the type strain Borrelia burgdorferi B31. The 41-kDa flagellin protein was present in all isolates, but the outer surface protein A (OspA) was absent in four isolates (HT15, HT2, HT20, and HT32). The molecular weights of the OspA proteins in six isolates were found to differ from one isolate to another. No two isolates examined had the same plasmid profile. These findings show the antigenic and genetic heterogeneity of the Japanese isolates, and some isolates are strikingly different from North American, European, and Asian strains.
Detection of immunoglobulin A in urine specimens from children with Campylobacter-associated diarrhea by a chemiluminescent indicator-based western immunoblot assay
Authors: Wu S J , Pazzaglia G , Haberberger R L , Oprandy J J , Sieckmann D G , Watts D M , Hayes C
Published: January 2020

A Western blot (immunoblot) assay was used to detect Campylobacter-specific immunoglobulin A in urine. Acute-phase urine samples from six children with Campylobacter diarrhea had titers ranging from 2 to 8. The highest titer was detected 4 days postonset. Campylobacter-specific immunoglobulin A was undetectable in the paired convalescent-phase specimens and urine samples from three control children.
Bacterial clump formation at the surface of liquid culture as a rapid test for identification of enteroaggregative Escherichia coli
Authors: Albert M J , Qadri F , Haque A , Bhuiyan N A
Published: January 2020

Forty-one strains of enteroaggregative Escherichia coli formed clumps visible as a scum at the surface of a Mueller-Hinton broth shaker culture. Sixty-one control strains of E. coli did not. Scum formation is a simple, rapid, and inexpensive test for the identification of enteroaggregative E. coli.
Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica
Authors: Flores B M , Reed S L , Ravdin J I , Torian B E
Published: January 2020

The 29-kDa peripheral membrane protein of Entamoeba histolytica has recently been demonstrated to have epitopes on pathogenic clinical isolates which were not detected by monoclonal antibodies on nonpathogenic isolates. To analyze the serological response to this protein, we tested 93 serum specimens (from 33 patients with amebic liver abscess, 7 patients with colitis, 2 patients with ameboma, 18 individuals harboring a nonpathogenic zymodeme strain, 10 healthy Mexican migrant workers, and 23 healthy controls) by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified native or recombinant protein. When tested by ELISA with the native antigen, 79% (26 of 33) of the serum specimens from patients with amebic liver abscess, 4 of 9 serum specimens from symptomatic patients with colitis or ameboma, and serum from one migrant worker were positive. None of the 18 subjects harboring a nonpathogenic strain or 23 control individuals were seropositive to the native antigen (sensitivity, 71%; specificity, 98%). Of 30 serum specimens from patients with amebic liver abscess tested with recombinant antigen, 27 were seropositive (90%). In addition, six patients with colitis or ameboma and two individuals who harbored a nonpathogenic strain were seropositive to the recombinant antigen. One healthy Mexican migrant worker tested positive by both ELISAs (sensitivity, 87%; specificity, 94%). Immunoblotting of 51 serum specimens to sodium dodecyl sulfate-denatured native 29-kDa protein was less sensitive (65%) than ELISA in detecting serum antibodies to the antigen. These results suggest a similar antibody response to native and recombinant antigens (r = 0.86) and support the potential utility of a quantitative assay with defined recombinant antigen for the serodiagnosis of invasive amebiasis in nonendemic areas in conjunction with other diagnostic tools.
Characterization of Brevibacterium spp. from clinical specimens
Authors: Gruner E , Pfyffer G E , von Graevenitz A
Published: January 2020

Nonfermenting coryneform bacteria identified as Brevibacterium spp. were isolated from routine clinical specimens. Four strains were derived from peritoneal fluid and has presumably been involved in the pathogenesis of continuous ambulatory peritoneal dialysis peritonitis. Another five isolates most probably represented skin contaminants. Cell wall and lipid analyses confirmed the genus identification. Strains in this taxon are difficult to distinguish from other biochemically inactive and nonmotile coryneform species but show characteristics cellular fatty acid profiles. In vitro susceptibilities to commonly used antibiotics were determined.