bluuebunny/crossref_metadata_2025 huggingface dataset:
Biologically distinct subtypes of Mycobacterium avium differ in possession of insertion sequence IS901
Authors: Kunze Z M , Portaels F , McFadden J J
Published: January 2020

Mycobacterium avium causes disease, principally tuberculosis in immunocompromised individuals. It is the most frequent cause of disseminated infections in AIDS patients in the West. The pathogen is also associated with disease in animals, chiefly birds and livestock, and may be isolated from environmental samples such as soil and water. Analysis of strains of M. avium isolated from clinical, veterinary, and environmental sources for the presence of the mycobacterial insertion sequences IS900 and IS901 demonstrates the specific association of IS901 to animal pathogenic M. avium strains. In contrast, most clinical M. avium strains and all AIDS-derived strains examined so far lacked IS901. Significant differences in the plasmid contents and serotypes of strains with and without IS901 were also found. We therefore suggest that the presence of IS901 divides M. avium into two clearly distinct subtypes with differing host range, virulence, plasmid possession, and serotyping antigens. By using DNA sequence data from IS901 and M. avium DNA, a set of polymerase chain reactions were developed for the specific detection and differentiation of these subtypes.
Antimicrobial susceptibility patterns of common and unusual species of enterococci causing infections in the United States. Enterococcal Study Group
Authors: Gordon S , Swenson J M , Hill B C , Pigott N E , Facklam R R , Cooksey R C , Thornsberry C , Jarvis W R , Tenover F C
Published: January 2020

We collected 705 isolates of enterococci (1 per patient) from cultures of a variety of anatomic sites from patients at eight tertiary-care hospitals in six geographic regions of the United States. A total of 632 (90%) Enterococcus faecalis, 58 (8%) E. faecium, 5 E. gallinarum, 4 E. avium, 3 E. casseliflavus, 1 E. raffinosus, and 1 E. hirae isolate and 1 biochemical variant of E. faecalis were identified; 606 (86%) of these isolates were associated with clinical infections. The most common sites of isolation were the urinary tract (402 [57%]), nonsurgical wounds (94 [13%]), the bloodstream (74 [10%]), and surgical wounds (62 [9%]). High-level resistance to gentamicin or streptomycin or both was detected in 265 (38%) of the isolates. We identified two E. faecalis isolates resistant to vancomycin (MICs, 32 and 128 micrograms/ml) and 11 beta-lactamase-producing E. faecalis isolates. E. faecium isolates were significantly more resistant than E. faecalis isolates to penicillin, ampicillin, piperacillin, imipenem, and ciprofloxacin (P less than 0.001). The MICs for the 15 non-E. faecalis, non-E. faecium enterococci indicated variable resistance to ciprofloxacin and the penicillins. Antimicrobial susceptibility patterns vary among species of enterococci, and these organisms, while commonly resistant to high-level aminoglycosides, can also acquire resistance to vancomycin or the ability to produce beta-lactamase. Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.
Detection of lipoarabinomannan as a diagnostic test for tuberculosis
Authors: Sada E , Aguilar D , Torres M , Herrera T
Published: January 2020

A coagglutination technique was established for the detection of lipoarabinomannan of Mycobacterium tuberculosis in human serum samples and evaluated for its utility in the diagnosis of tuberculosis at the Instituto Nacional de Enfermedades Respiratorias in Mexico City. The test had a sensitivity of 88% in patients with sputum-smear-positive active pulmonary tuberculosis. The sensitivity in patients with active pulmonary tuberculosis negative for acid-fast bacilli in sputum was 67%. Less favorable results were obtained for patients with AIDS and tuberculosis, with a sensitivity of 57%. The specificity in control patients with lung diseases different from tuberculosis and in healthy subjects was 100%. The positive predictive value was 100%, and the negative predictive value for patients with sputum-positive active pulmonary tuberculosis was 97%. The results of this study suggest that the detection of lipoarabinomannan is an accurate test for the diagnosis of pulmonary tuberculosis.
Detection of Coxiella burnetti by DNA amplification using polymerase chain reaction
Authors: Stein A , Raoult D
Published: January 2020

The polymerase chain reaction (PCR) was used for the detection of Coxiella burnetti, an obligate intracellular bacterium and the etiologic agent of Q fever. A pair of primers derived from the C. burnetii superoxide dismutase gene served to amplify a targeted 257-bp fragment of genomic DNA. These primers were chosen on the basis of GenBank analysis, G + C ratio, and absence of secondary structure. This technique allowed the detection of as few as 10 C. burnetii organisms. C. burnetti was detected in tissue culture and in specimens from patients (heart valves). In all, 8 reference isolates and 22 new isolates of C. burnetii from France were successfully amplified. No amplification products were found when PCR was performed with 25 bacterial species that had been isolated in a clinical laboratory from patients with clinically similar infections. Amplification products of C. burnetii were confirmed by restriction enzyme digestion and dot blot hybridization. The method used here, a combination of PCR and restriction analysis, is a faster and more sensitive assay for C. burnetii than standard culture techniques.
Murine Pneumocystis carinii adherence to vertical monolayers of cultured mink lung cells (MiCl1)
Authors: Garner R E , Walker A N , Horst M N
Published: January 2020

We describe a method for adherence and culture of murine Pneumocystis carinii in mink lung cells (MiCl1) grown on vertical supports. The vertical cultures were infected with P. carinii; the surrounding medium and inoculum were stirred to ensure circulation and contact with MiCl1 cells. When compared with conventional horizontal culture, the vertical method offers a more suitable system for assessing P. carinii adherence. This approach has proved suitable for quantitative evaluation of P. carinii adherence to MiCl1 cells in the presence of inhibitors.
Endocarditis caused by a group B Streptococcus strain, type III, in a nonencapsulated phase
Authors: Sellin M , Linderholm M , Norgren M , Håkansson S
Published: January 2020

A nontypeable blood isolate of group B streptococci (GBS) from a patient with endocarditis is suggested to be the nonencapsulated phase of a GBS strain, type III. From the original high-density isolate, a low-density, encapsulated phase was selected by Percoll gradient centrifugation. This phenomenon should be considered before a GBS strain is classified as truly nontypeable.
Evaluation of nonradioactive DNA probes for identification of mycobacteria
Authors: Lebrun L , Espinasse F , Poveda J D , Vincent-Levy-Frebault V
Published: January 2020

Commercial chemiluminescent DNA probes (Accuprobe; Gen-Probe, San Diego, Calif.) for the identification of Mycobacterium tuberculosis (MTB) complex, M. avium complex (MAC), M. gordonae, and M. kansasii were evaluated with 134 clinical isolates. These included 36 MTB complex, 40 MAC, 27 M. gordonae, 9 M. kansasii, and 22 Mycobacterium spp. The specificity was 100% for the four probes. The sensitivity was 100% for the MTB complex and M. gordonae probes and 95.2% for the MAC probe. Five of the nine M. kansasii isolates tested were not detected with the probe.
Paecilomyces lilacinus catheter-related fungemia in an immunocompromised pediatric patient
Authors: Tan T Q , Ogden A K , Tillman J , Demmler G J , Rinaldi M G
Published: January 2020

Paecilomyces lilacinus catheter-related fungemia in an immunocompromised child is reported. The presence of a central venous catheter and the patient's immunocompromised status were felt to be predisposing factors for this unusual infection. To our knowledge, this is the first description of P. lilacinus catheter-related fungemia, and our patient may be the youngest reported patient with this mycosis who was cured.
Haemophilus influenzae is frequently detected with monoclonal antibody 8BD9 in sputum samples from patients with cystic fibrosis
Authors: Möller L V , Ruijs G J , Heijerman H G , Dankert J , van Alphen L
Published: January 2020

To determine the frequency of Haemophilus influenzae in sputum from patients with cystic fibrosis (CF), 477 sputum samples obtained from 86 CF patients were analyzed by standard culture and by the in situ immunoperoxidase staining technique with monoclonal antibody 8BD9. H. influenzae was isolated from 109 sputum samples (23%) from 45 patients (52%) and detected by immunoperoxidase staining in 175 sputum samples (37%) obtained from 63 patients (73%). The results of this study demonstrate the frequent presence of H. influenzae in sputum samples from CF patients.
Bilophila wadsworthia bacteremia in two patients with hepatic abscesses
Authors: Kasten M J , Rosenblatt J E , Gustafson D R
Published: January 2020

Bilophila wadsworthia, a recently described anaerobic gram-negative bacillus, was isolated from blood cultures of two patients with liver abscesses. These isolates exhibited the expected phenotypic characteristics, and identification was confirmed by cell wall fatty acid analysis by gas-liquid chromatography. This is the first documentation of bacteremia due to B. wadsworthia.
Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin
Authors: Yam W C , Lung M L , Ng M H
Published: January 2020

The effectiveness of a latex agglutination assay kit for the detection of Escherichia coli heat-labile toxin and cholera toxin was determined for the identification of natural isolates of the corresponding enteric pathogens in Southeast Asia. By selection of the appropriate culture media, the sensitivity of the assay was improved from 90.6% (for the detection of heat-labile toxin) and 75% (for the detection of cholera toxin) to 100%, and the results were confirmed with bioassays and DNA hybridization assays for both clinical and environmental isolates.
Value of a DNA probe assay (Gen-Probe) compared with that of culture for diagnosis of gonococcal infection
Authors: Vlaspolder F , Mutsaers J A , Blog F , Notowicz A
Published: January 2020

The Gen-Probe PACE 2 system for Neisseria gonorrhoeae (GP), which uses a chemiluminescently labeled DNA probe, was compared with conventional culture as the method of reference. A total of 1,750 specimens were collected from 496 females and 623 males visiting the outpatient clinic of the Sexually Transmitted Diseases Department of the Westeinde Hospital, The Hague, The Netherlands, during the year 1991. The prevalences of gonorrhea culture-positive men and women were 14.9 and 7.7%, respectively. The overall positive rate was 8.7%. Sensitivity, specificity, and positive and negative predictive values of GP were 97.1, 99.1, 90.6, and 99.8%, respectively. A total of 12 of 13 patients with positive GP results and negative cultures may have had a gonococcal infection, a conclusion based on clinical symptoms, positive methylene blue smears, and high relative light unit ratios. The DNA probe test can be useful as a suitable screening and diagnostic test for gonorrheal infection in men and women. An advantage of using this DNA probe technique is that simultaneous testing for Chlamydia trachomatis of the same specimen is possible. We also examined whether (all) rRNA had disappeared after adequate treatment for gonococcal and/or chlamydial infection in 30 patients. None of those positive patients showed a positive result in the DNA probe assay after treatment.
Epidemiological fingerprinting of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments
Authors: Haertl R , Bandlow G
Published: January 2020

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.
Detection of Francisella tularensis in blood by polymerase chain reaction
Authors: Long G W , Oprandy J J , Narayanan R B , Fortier A H , Porter K R , Nacy C A
Published: January 2020

We developed a polymerase chain reaction-based assay for Francisella tularensis which we evaluated by using spiked blood samples and experimentally infected mice. The assay detected both type A and type B F. tularensis at levels equivalent to one CFU/microliter of spiked blood. Results from polymerase chain reaction-based assay of limiting dilutions of blood from mice infected with the live vaccine strain agreed closely with results from blood culture.
Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae O1 strains
Authors: Olsvik O , Wahlberg J , Petterson B , Uhlén M , Popovic T , Wachsmuth I K , Fields P I
Published: January 2020

Cholera toxin is the principal factor causing the profuse intestinal fluid secretion that is characteristic of cholera. The DNA sequences of the cholera toxin subunit B structural genes from 45 Vibrio cholerae O1 strains isolated in 29 countries over a period of 70 years were determined by automated DNA sequencing of polymerase chain reaction-generated amplicons. Three types of cholera toxin B subunit gene (ctxB) were identified. Genotype 1 was found in strains of classical biotype worldwide and El Tor biotype strains associated with the U.S. Gulf Coast, genotype 2 was found in El Tor biotype strains from Australia, and genotype 3 was found in El Tor biotype strains from the seventh pandemic and the recent Latin American epidemic. All base changes correspond to an amino acid substitution in the B subunit of the cholera toxin. Heterogenicity in the B subunit could have implications for vaccine development and diagnostic tests for cholera toxin and antitoxin. We conclude that this technology provides timely and potentially useful epidemiological information.
Comparison of nasopharyngeal aspirates with swabs for culture of Bordetella pertussis
Authors: Hallander H O , Reizenstein E , Renemar B , Rasmuson G , Mardin L , Olin P
Published: January 2020

Nasopharyngeal samples were collected from 117 children by aspiration from one nostril and by swab from the other one and cultured for Bordetella pertussis. Among 33 culture-positive specimens, there were 30 positive aspirates and 26 positive swab specimens. Aspirates are easily divided and saved for investigations with other assays which may further improve diagnostic sensitivity. As the aspiration technique was also preferred by nurses and parents, it was recommended and chosen for a planned pertussis vaccine efficacy trial to take place in Sweden from 1992 to 1995.
Prevalence of dental caries in 4- to 5-year-old children partly explained by presence of salivary mutans streptococci
Authors: Granath L , Cleaton-Jones P , Fatti L P , Grossman E S
Published: January 2020

The correlation between dental caries and the number of oral mutans group streptococci (ms) present has been shown to be weak. The aim of this investigation was to study associations between caries experience (decayed, missing, and filled surfaces [dmfs]) and the number of ms in stimulated saliva, with emphasis on the level of disease and the confounding effect of regular intake of sweets, the presence of salivary lactobacilli, and oral hygiene. In some 2,700 4- to 5-year-old South African children of different ethnic origins, caries was diagnosed on the basis of World Health Organization criteria and saliva samples were analyzed for ms after cultivation on mitis salivarius-bacitracin agar and for lactobacilli by using the Dentocult kit. Oral hygiene was scored on the basis of the Greene and Vermillion simplified debris index, while data on intake of sweets were derived from extensive interviews. Pearson's coefficient of correlation was computed, and multiple regression analysis was performed to correct for confounding factors. The distribution of the children in the eight caries classes was strongly associated with the ms class (P < 0.001), with those in the lower ms classes generally having low dmfs scores and those in the higher ms classes having dmfs scores distributed over the whole range. The r value for the two variables was 0.25 for the total material; this was reduced to 0.18 by correction for confounding factors. The corresponding values for children with caries were 0.21 and 0.17, for those in the 1 to 6 dmfs interval they were 0.07 and 0.03, and for those in the 7 to 81 dmfs interval they were 0.16 and 0.14. The data imply that the explanatory values for ms, those for the lower caries interval not counted, ranged from 6 to 2%. The unexpected results for children with caries might be due to their distribution pattern. It is concluded that there is a need for reevaluation of ms as a risk factor in dental caries.
Ribotyping of Pseudomonas aeruginosa: discriminatory power and usefulness as a tool for epidemiological studies
Authors: Blanc D S , Siegrist H H , Sahli R , Francioli P
Published: January 2020

Restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) of Pseudomonas aeruginosa was evaluated as a tool for epidemiological purposes. Fifty-five epidemiologically unrelated isolates from three geographic areas of Switzerland and 11 isolates obtained during an outbreak of P. aeruginosa infections in a burn unit were typed by this method. Typeability and reproducibility of the method reached 100%. With four selected restriction enzymes (BamHI, ClaI, EcoRI, and PstI), the 55 unrelated isolates could be classified into 33 ribotypes. To assess the value of this method for the interpretation of epidemiological data, we calculated an index of discrimination (ID) which takes into consideration both the number of types defined by the typing method and their relative frequencies. Our ribotyping system obtained a high ID of 0.958 with only four restriction enzymes, comparing well with other different typing schemes for which ID values could be calculated from published data. All clinical isolates of the outbreak belonged to the same ribotype, whereas environmental isolates, initially thought to be the source of the epidemic, belonged to a different ribotype. Thus, the typeability, reproducibility, and discriminatory power of our method as well as its value established in an epidemiological investigation were found to be appropriate for further epidemiological studies of P. aeruginosa.
Detection of the pathogenic parasite Toxoplasma gondii by specific amplification of ribosomal sequences using comultiplex polymerase chain reaction
Authors: Guay J M , Dubois D , Morency M J , Gagnon S , Mercier J , Levesque R C
Published: January 2020

Amplification of DNA sequences from ribosomal DNA (rDNA) was tested as a specific and sensitive method for the detection of small numbers of Toxoplasma gondii tachyzoite cells. We applied the polymerase chain reaction (PCR) on the basis of detection of the 110-fold repetitive rDNA as a target by using (i) DNA sequences within the small ribosomal subunit known to be universal and conserved in all eukaryotes and (ii) small ribosomal subunit and intergenic spacer rDNA sequences known to be T. gondii species specific. The level of sensitivity obtained from a crude cell lysate allowed the detection of as few as one parasite visualized directly as a specific PCR product in agarose gels. By using a combination of universal and T. gondii species-specific primers, we propose a comultiplex-based PCR approach as a new diagnostic tool. The combination of sensitivity, specificity, and built-in positive and negative PCR controls should make detection of the rDNA sequences by comultiplex PCR a useful clinical test for the diagnosis of toxoplasmosis and for epidemiological studies. Finally, the idea of a built-in positive control to support or counter the T. gondii-specific PCR result is novel and is a notable advance.
Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction
Authors: Olsson M , Elvin K , Löfdahl S , Linder E
Published: January 2020

A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii.
Use of ribotyping for characterization of Salmonella serotypes
Authors: Esteban E , Snipes K , Hird D , Kasten R , Kinde H
Published: January 2020

Forty-five isolates of Salmonella serotype reading, 20 isolates of Salmonella serotype senftenberg, and 56 isolates of Salmonella serotype typhimurium from domestic and wild animals were characterized genotypically to differentiate within serotypes for epidemiologic studies. The genotypic method of characterization used was ribotyping, a method for highlighting highly conserved rRNA genes and associated sequences. Isolates were obtained from diverse geographic sources (farms located in Fresno, Sonoma, Stanislaus, and Yolo counties) as well as different hosts (avian, equine, bovine, murine, and environmental) during a period of 8 months. Within a given serotype, ribotying was able to establish subclassifications (ribotypes) that grouped isolates by a common source regardless of host or geographic origin. There were four distinct ribosomal banding patterns observed for Salmonella serotype reading, six were observed for Salmonella serotype senftenberg, and two were observed for Salmonella serotype typhimurium.
Enterobacter taylorae, a new opportunistic pathogen: report of four cases
Authors: Rubinstien E M , Klevjer-Anderson P , Smith C A , Drouin M T , Patterson J E
Published: January 2020

Severe nosocomial infections due to Enterobacter taylorae (formerly known as CDC Enteric Group 19) are described in four patients. Unlike most members of the Enterobacter genus, the isolates were not susceptible to penicillins or cephalosporins. Restriction endonuclease analysis of E. taylorae DNA obtained from three patients identified two distinct strains. One strain was found in two patients, suggesting a common source which we were not able to identify. We postulate that in patients harboring E. taylorae, the combination of cephalosporin therapy and instrumentation enables this organism to become an opportunistic pathogen.
Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR
Authors: Gretch D R , Wilson J J , Carithers R L , dela Rosa C , Han J H , Corey L
Published: January 2020

We evaluated a new hepatitis C virus RNA assay based on one-stage PCR followed by liquid hybridization with an oligonucleotide probe and compared it with nested-set PCR. The one-stage and nested-set PCR assays had identical sensitivities in analytical experiments and showed 100% concordance when clinical specimens were used. One-stage PCR may be less prone to contamination than nested-set PCR.
Identification of a clone of Escherichia coli O103:H2 as a potential agent of hemolytic-uremic syndrome in France
Authors: Mariani-Kurkdjian P , Denamur E , Milon A , Picard B , Cave H , Lambert-Zechovsky N , Loirat C , Goullet P , Sansonetti P J , Elion J
Published: January 2020

In a French multicenter study, six verocytotoxin-producing Escherichia coli strains were isolated from the stools of 6 of 69 children suffering from hemolytic-uremic syndrome. All strains belonged to serotype O103:H2, a serotype commonly associated with diarrhea in weaned rabbits in France. To determine whether the strains from humans and rabbits were genetically related, they were compared by analyzing their esterase electropherotypes and the restriction fragment length polymorphisms of the ribosomal DNA regions. A common clonal origin of these pathogenic strains was suggested by their identical esterase electropherotypes and their identical ribotypes, in addition to their identical serotypes. However, strains from humans, which are cytotoxic for HeLa cells through the production of verocytotoxin type 1, do not show adhesion in vitro to HeLa 229 cells and cannot infect rabbits. On the other hand, strains from rabbits do not carry the verocytotoxin type 1 gene, are not cytotoxic for Hela cells, and adhere to ileal villi and HeLa 229 cells because of the expression of their 32-kDa adhesin. Our results therefore identify a clone of verocytotoxin-producing E. coli O103:H2 as a potential agent of hemolytic uremic syndrome in France. They further suggest that clones from humans and rabbits probably have a common origin but that adaptation to the two species occurred by different mechanisms. Thus, they eliminate the hypothesis that the species is horizontally transmitted between rabbits and humans.
Detection of parvovirus B19 in donated blood: a model system for screening by polymerase chain reaction
Authors: McOmish F , Yap P L , Jordan A , Hart H , Cohen B J , Simmonds P
Published: January 2020

A highly sensitive and rapid method for routinely screening large numbers of donated blood units for parvovirus B19 by the polymerase chain reaction (PCR) was developed. Over a 3-month trial period in Edinburgh, B19 DNA was detected in 6 of 20,000 consecutive units of blood (0.03%), in concentrations ranging from 2.4 x 10(4) to 5 x 10(10) copies of viral DNA per ml. Seroconversion for B19-specific immunoglobulin M and immunoglobulin G and disappearance of circulating B19 DNA occurred in the interval between donation and recall in four of the five implicated donors who could be recalled. B19 DNA was detected in 18 of 27 separate batches of non-heat-treated factor VIII and IX concentrate manufactured from donated plasma unscreened for B19 DNA. Dry-heat treatment at 80 degrees C for 72 h reduced but did not always eliminate detectable B19 from factor VIII concentrates, consistent with recent observations that current methods for virus inactivation during blood product manufacture are insufficient to entirely eliminate B19 infectivity. The methods developed in this study for PCR screening could be applied routinely to prevent transfusion of B19 in blood and blood products and could play an important role in the prevention of iatrogenic transmission of infection. PCR screening could also be used for detection and exclusion of a range of other transmission-associated viruses for which current serological detection methods are only partially effective.
Rapid, simple method for typing isolates of Mycobacterium tuberculosis by using the polymerase chain reaction
Authors: Ross B C , Dwyer B
Published: January 2020

To develop a molecular typing method for Mycobacterium tuberculosis based on the polymerase chain reaction, oligonucleotide primers were designed to the ends of the insertion sequence IS6110 in an attempt to amplify DNA between clusters of this element on the genome. Although in many strains the copy number of this element is low and is distributed throughout the genome, most strains examined produced a banding pattern which varied between isolates including strains with one copy of IS6110. With strains isolated from patients in epidemiologic clusters of tuberculosis, the banding patterns were similar within each cluster but distinct from those in strains from different clusters. Similarly, multiple isolates from the same patient yielded a consistent banding pattern. Sequencing of four polymerase chain reaction products revealed that amplification was occurring between copies of IS6110 in two of the products and from a single copy of IS6110 to a nonspecific priming site in the other two. This technique provides a rapid and simple means of typing M. tuberculosis isolates for epidemiologic studies.
Purification of the 115-kilodalton exoantigen of Cryptococcus neoformans and its recognition by immune sera
Authors: Hamilton A J , Goodley J
Published: January 2020

A 115-kDa exoantigen produced by Cryptococcus neoformans recognized by the previously described murine monoclonal antibody 7C9 has been purified from culture filtrate by a combination of membrane ultrafiltration, isoelectric focusing, and preparative gel electrophoresis. It is produced in late-log-phase cultures and is present in greater amounts in cultures grown at 25 degrees C than in those grown at 37 degrees C. Recognition of the antigen by 7C9 on immunoblots is abolished by the proteolytic enzymes papain and trypsin. The antigen is a glycoprotein bearing N-linked oligosaccharides, of which mannose is an important constituent. It does not appear to have proteolytic activity and is acidic, with a pI of 3 to 3.2. Its relationship to previously described C. neoformans mannoprotein is unclear since 7C9 shows only very weak cross-reactivity with a purified sample of the latter. Sera from patients infected with C. neoformans exhibited strong recognition of the glycoprotein as shown by immunoenzyme development of Western immunoblots, indicating its possible significance as a marker of disease.
Comparison of isolation methods for the recovery of Bordetella bronchiseptica and Pasteurella multocida from the nasal cavities of piglets
Authors: Lariviere S , Leblanc L , Mittal K R , Martineau G P
Published: January 2020

Nasal swabs from 241 piglets from 12 herds with clinical atrophic rhinitis and 283 piglets from 14 herds without clinical atrophic rhinitis were examined for the presence of Bordetella bronchiseptica and/or Pasteurella multocida. For B. bronchiseptica, swabs were streaked on three selective media. Blood agar supplemented with cephalexin was the most satisfactory selective culture medium for the isolation of B. bronchiseptica. For P. multocida, swabs were also streaked on three selective media. Mice were also used for isolation of P. multocida from the nasal cavities of pigs. The mouse inoculation test was not found to be the definitive test for the isolation of P. multocida. A significant number of P. multocida strains were avirulent in the mouse model. The modified Knight medium (without potassium tellurite) was the best single method for isolating P. multocida. However, a combination of mouse passage and direct culture on selective media increased the rate of isolation. There was no marked difference in the prevalence of B. bronchiseptica or P. multocida in swine herds with or without clinical atrophic rhinitis. Both capsular types A and D were present in the nasal cavities of the pigs with or without clinical atrophic rhinitis.
Immunoglobulin G subclass responses against the structural components of Puumala virus
Authors: Lundkvist A , Björsten S , Niklasson B
Published: January 2020

Sera from sequentially bled nephropathia epidemica patients (acute-phase, convalescent-phase, and 2-year sera) and sera from 10 to 20 years postinfection were examined by immunoglobulin G (IgG) subclass-specific ELISAs for reactivities against each of the Puumala virus structural proteins (N, G1, and G2). IgG1 was found to be the dominating antiviral subclass, and most of the patients had IgG1 directed to all three structural proteins at all times with a continuous increase over time. IgG2 to the three viral proteins was detected in very low amounts in only a few of the samples. All of the serially bled patients developed IgG3 against each of the proteins, but the responses against the different viral components varied; anti-N- and anti-G1-specific IgG3 showed a peak in the convalescent-phase samples, while the anti-G2 IgG3 response was highest in the acute-phase sera. Eight of ten serially bled patients developed specific IgG4. The IgG4 responses showed similar patterns against all three viral proteins with very low optical density values in the acute- and convalescent-phase samples, followed by higher optical densities in the sera drawn 2 years after infection. Seven of nine 10-year sera contained virus-specific IgG4. These results support the theory concerning the persistence of Puumala virus or viral antigens several years after infection.
Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen
Authors: Gottstein B , Jacquier P , Bresson-Hadni S , Eckert J
Published: January 2020

Alveolar echinococcosis (AE) in humans is generally a fatal disease when not diagnosed early enough to provide curative treatment such as radical surgery. Immunodiagnosis for early detection of AE was improved by the isolation of an affinity-purified metacestode Em2 antigen and by the synthesis of recombinant Echinococcus multilocularis antigen II/3-10. Both antigens were individually assessed by enzyme-linked immunosorbent assay (ELISA) and demonstrated high specificities and diagnostic sensitivities, although both missed approximately 4 to 11% of diagnostic cases of AE. To provide an optimal serodiagnostic test, we investigated the two purified antigens by using a test employing a mixture of both purified antigens (designated Em2plus antigen) in one assay. For comparative purposes, crude E. multilocularis and Echinococcus granulosus metacestode antigens were investigated as well. The Em2plus ELISA proved to be the optimal diagnostic test with the highest diagnostic sensitivity, 97%, in serum samples from 140 patients with AE and an overall specificity of 99% for infections due to other Echinococcus and non-Echinococcus parasites. The new test combination (Em2plus ELISA) is suggested for the serodiagnosis of AE in patients and for seroepidemiological surveys.
Rapid detection of poliovirus by reverse transcription and polymerase chain amplification: application for differentiation between poliovirus and nonpoliovirus enteroviruses
Authors: Abraham R , Chonmaitree T , McCombs J , Prabhakar B , Lo Verde P T , Ogra P L
Published: January 2020

This report describes a rapid method of detection of poliovirus from viral isolates of clinical specimens using a single set of primers selected from the conserved 5' noncoding region of the poliovirus genome. Of the 144 clinical viral isolates examined, 81 were positive for polioviruses and 50 were positive for nonpoliovirus enteroviruses by tissue culture neutralization and infectivity. All 81 (100%) of the viral isolates identified as poliovirus by tissue culture infectivity were also positive by polymerase chain reaction. Of 50 nonpoliovirus enterovirus isolates found to be negative for poliovirus by tissue culture neutralization and infectivity, 48 were also negative by polymerase chain reaction. The high sensitivity (100%) and specificity (96%) of the primer set indicate that this assay has potential clinical applicability in the diagnosis of nonpoliovirus enterovirus infection.
Evaluation of Gen-Probe's Histoplasma capsulatum and Cryptococcus neoformans AccuProbes
Authors: Huffnagle K E , Gander R M
Published: January 2020

Gen-Probe's DNA probes were evaluated for use in the identification of clinical isolates of Histoplasma capsulatum var. capsulatum and Cryptococcus neoformans. Ninety-five mould-phase fungi were probed, including 41 isolates of H. capsulatum var. capsulatum. Similarly, 98 yeasts, including 42 C. neoformans isolates, were examined by using the C. neoformans DNA probe. In the study, both probes demonstrated 100% specificity and 100% sensitivity. Their use in the clinical laboratory may significantly reduce the time required for definitive identification of fungi.
Effect of gamma irradiation on reactivity of rinderpest virus antigen with bovine immune serum in enzyme-linked immunosorbent assays and virus neutralization and indirect fluorescent-antibody tests
Authors: Saliki J T , Berninger M L , Torres A , House J A , Mebus C A , Dubovi E J
Published: January 2020

Gamma irradiation effectively inactivated gradient-purified rinderpest virus. Irradiated antigen and sera remained functional in enzyme-linked immunosorbent assays, virus neutralization tests, and indirect fluorescent-antibody tests. Irradiation, however, led to a dose-dependent decrease in reactivity, particularly significant (P < 0.05) when both reagents were irradiated. To avoid false-positive reactions, only one reagent (serum or antigen) may be irradiated.
Fatal pneumonia due to Serratia proteamaculans subsp. quinovora
Authors: Bollet C , Grimont P , Gainnier M , Geissler A , Sainty J M , De Micco P
Published: January 2020

Serratia proteamaculans subsp. quinovora was isolated from several samples (blood cultures, tracheal aspirates, pleural effusion) from a patient with pneumonia. This is the first clinical isolate and the first documented human infection caused by this organism.
Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses
Authors: Poutrel B , Sutra L
Published: January 2020

A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable.
Detection of feline immunodeficiency virus in saliva and plasma by cultivation and polymerase chain reaction
Authors: Matteucci D , Baldinotti F , Mazzetti P , Pistello M , Bandecchi P , Ghilarducci R , Poli A , Tozzini F , Bendinelli M
Published: January 2020

The rates of feline immunodeficiency virus (FIV) isolation from saliva, plasma, and peripheral blood mononuclear cells (PBMC) of infected cats were compared; isolation rates were 18, 14, and 81%, respectively, in naturally infected cats and 25, 57, and 100%, respectively, in experimentally infected animals. There was no obvious relationship between isolation rate and clinical stage or between isolation rate and the titer of neutralizing antibody in serum. Virus could be isolated from one salivary gland as early as 1 week postinfection and, on a more regular basis, starting at 3 weeks postinfection, when, however, most other tissues were also positive. Polymerase chain reaction analysis showed that FIV genomes are present in saliva and plasma more frequently than expected on the basis of isolation data. Saliva was also found to contain viral DNA, indicating that it may harbor virus-infected cells as well as free virus. The addition of plasma but not of saliva to PBMC cultures delayed FIV growth. Isolation from plasma may be hampered by FIV neutralizing antibody and by the cytotoxic activity of this fluid for the PBMC used as a cell substrate.
Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens
Authors: de Wit M Y , Douglas J T , McFadden J , Klatser P R
Published: January 2020

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19% of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.
Specific detection of toxigenic strains of Clostridium difficile in stool specimens
Authors: Gumerlock P H , Tang Y J , Weiss J B , Silva J
Published: January 2020

Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.
Diagnosis of invasive candidiasis by a dot immunobinding assay for Candida antigen detection
Authors: Reboli A C
Published: January 2020

A dot immunobinding assay which uses a polyclonal rabbit anti-Candida immunoglobulin G as the primary antibody and colloidal gold coated with goat anti-rabbit immunoglobulin G as the secondary antibody for the detection of Candida cytoplasmic antigens is described. It was able to detect as little as 1 ng of total Candida protein per ml when a cytoplasmic extract of Candida albicans was seeded into buffer and 10 ng/ml when the same extract was seeded into pooled human serum. Serial serum samples from four groups of patients were assayed for Candida antigen: (i) 22 patients with candidemia, (ii) 16 patients at high risk for invasive candidiasis, (iii) 3 patients with other deep mycoses, and (iv) 50 hospitalized patients at low risk for serious Candida infection. Of the 22 candidemic patients, 19 had invasive candidiasis and 3 had transient candidemia. Antigenemia was detected in 16 of the 19 patients with invasive candidiasis (including patients with C. albicans, Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis) and in 4 of 16 patients at high risk for invasive candidiasis. There was no detectable antigen in 12 high-risk control patients, 3 patients with transient candidemia, 3 patients with other deep mycoses, and 50 relatively low-risk patients. The sensitivity for detecting invasive disease in candidemic patients and specificity for all patients studied were 84.2 and 94.4%, respectively. The positive predictive value was 80%; the negative predictive value was 95.7%. The sensitivity for neutropenic patients with invasive disease was 85.7%. This assay is rapid and accurate and appears to be useful in identifying candidemic patients with invasive candidiasis.
Molecular typing of Pseudomonas pseudomallei: restriction fragment length polymorphisms of rRNA genes
Authors: Lew A E , Desmarchelier P M
Published: January 2020

The aim of this study was to develop a typing scheme for Pseudomonas pseudomallei by comparison of patterns of restriction fragment length polymorphisms in rRNA genes (ribotyping). BamHI restriction digests of 100 isolates from various animal (34), human (58), and environmental (6) sources, including six reference strains, were hybridized to Escherichia coli 16S and 23S rRNAs. A chemiluminescent labelling and detection system was used to visualize bands. On the basis of patterns, the strains were classified into 22 different groups, with the largest containing 29 isolates. While most of the ribotypes were not exclusive to a particular source, some ribotypes were restricted to a particular geographic area or to either a human or a particular animal species. Application of the typing scheme to isolates of four independent outbreaks among animals showed that certain ribotypes predominated. The study demonstrated ribotyping to be a useful tool in epidemiological investigations of melioidosis.
Characterization of a nested polymerase chain reaction assay for detection of parvovirus B19
Authors: Patou G , Pillay D , Myint S , Pattison J
Published: January 2020

The characterization and application of a nested polymerase chain reaction (PCR) assay for the detection of human parvovirus B19 DNA is described. The assay was evaluated with 149 diagnostic serum samples (collected up to 150 days after the onset of symptoms) previously tested by dot blot hybridization for B19 DNA and by class-specific capture radioimmunoassays for the detection of B19 immunoglobulin M (IgM) and IgG. B19 DNA was detectable by the PCR in 70% of the sera. There was a statistically significant association between the detection of B19 DNA by PCR and high B19 IgM values (P < 0.005), low B19 IgG values (P < 0.05), and a short interval between onset of symptoms and serum collection (P < 0.005). Serial serum samples, throat swabs, and peripheral blood mononuclear cells collected from 10 individuals during an outbreak of parvovirus B19 were also tested by the nested PCR. B19 DNA was detectable in the throat swabs at the time of the clinical illness and in the peripheral blood mononuclear cell fraction up to the end point of the study 6 months after infection. The location of the B19 DNA could not be determined in cytocentrifuge preparations of peripheral blood mononuclear cells with nonisotopic in situ hybridization and immunolabelling.