bluuebunny/crossref_metadata_2025 huggingface dataset:
Multicenter clinical evaluation of a continuous monitoring blood culture system using fluorescent-sensor technology (BACTEC 9240)
Authors: Nolte F S , Williams J M , Jerris R C , Morello J A , Leitch C D , Matushek S , Schwabe L D , Dorigan F , Kocka F E
Published: January 2020

The BACTEC 9240 (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) is a new continuous-monitoring blood culture system that uses internal, fluorescent-CO2 sensors. In a multicenter clinical trial, organism yield and times to detection with the prototype BACTEC 9240 system were compared with those of the BACTEC NR 660 system. Equal volumes of blood were inoculated into the bottles included in the study blood culture sets (aerobic and anaerobic 9240 and NR6A and NR7A bottles). A total of 9,391 aerobic and 8,951 anaerobic bottle pairs were inoculated with 9,801 blood specimens. A total of 587 clinically significant positive blood cultures and 415 cases of sepsis were studied. The standard 9240 aerobic bottle detected significantly more Staphylococcus aureus (P < 0.05), coagulase-negative staphylococci (P < 0.01), and total microorganisms (P < 0.001) than the NR6A bottle. The standard 9240 anaerobic bottle detected significantly more coagulase-negative staphylococci (P < 0.001), members of the family Enterobacteriaceae (P < 0.01), and total microorganisms (P < 0.001) than the NR7A bottle. A total of 420 positive cultures were detected in both systems; for 284, the time to detection was equivalent with both systems (within 12 h); for 123, the 9240 system was faster; and for 13, the NR 660 system was faster (P < 0.001). The average times to detection for the 9240 and the NR 660 systems were 20.2 and 27.5 h, respectively. Ninety-nine cultures were positive only in the 9240 system, and 68 cultures were positive only in the NR 660 system (P < 0.02). The 9240 system also detected significantly more episodes of bacteremia (P < 0.001). The false-positive rates for the 9240 and NR 660 systems were 2.2 and 2.3%, respectively. The false-negative rates for the two systems after 5 days of incubation did not differ significantly. The contamination rates for the 9240 and NR 660 systems were 1.9 and 1.5%, respectively (P < 0.05). In conclusion, the prototype 9240 system detected more clinically significant positive blood cultures and did so sooner than the NR 660 system, with the additional advantages of full automation, continuous monitoring, and noninvasive sampling.
Characterization of monoclonal antibodies against putative colonization factors of enterotoxigenic Escherichia coli and their use in an epidemiological study
Authors: Viboud G I , Binsztein N , Svennerholm A M
Published: January 2020

Monoclonal antibodies (MAbs) against five putative colonization factors (PCFs), i.e., colonization factor antigen (CFA)/III, coli surface antigen (CS)7 and CS17, PCFO159, and PCFO166 of enterotoxigenic Escherichia coli (ETEC), were produced. Hybridomas (one each) producing specific antibodies against the respective PCFs were selected. All the MAbs reacted with the corresponding fimbriae but not with CFA/I, CFA/II, or CFA/IV or the heterologous PCFs in bacterial agglutination and enzyme-linked immunosorbent assays (ELISAs). In immunoelectron microscopy these MAbs bound along the fimbriae, and they also reacted with the corresponding subunits in immunoblots. The five MAbs were used to evaluate the prevalence of CFA/III, CS7, CS17, PCFO159, and PCFO166 in ETEC strains isolated from children with diarrhea in Argentina. One hundred five ETEC isolates negative for CFA/I, CFA/II, and CFA/IV were tested in slide agglutination or in a dot blot test for spontaneously agglutinating strains; positive results were confirmed by inhibition ELISAs. It was found that 27% of the CFA-negative ETEC strains carried one of the PCFs. The sensitivity of slide agglutination with these MAbs was similar to that with specific polyclonal antisera; however, the specificity was higher. PCFO166 was found in 9.5% of the strains tested, mainly in ETEC of serogroup O78 producing heat-stable toxin alone. CS17 and CS7 were identified in 6.7 and 5.7%, respectively, of strains producing heat-labile toxin only, most of which belonged to serogroup O114. PCFO159 was found in 3.8% of the isolates tested, whereas CFA/III was detected in only one ETEC strain.
Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach
Authors: Katz J B , Ridpath J F , Bolin S R
Published: January 2020

Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) are closely related pestiviruses. BVDV and BDV are found worldwide but seldom cause disease in swine. In contrast, HCV has been successfully eradicated from swine in several nations but poses a potentially devastating threat to them because of its great virulence. Rapid differential diagnosis of HCV from BVDV and BDV infections in swine is vital for detection of the possible reintroduction of HCV into national herds from which it has been eradicated. Nested polymerase chain reactions (PCRs) for each of two pestiviral genomic segments are described. Amplification of the relatively conserved 5' genomic terminus identified 59 of 61 HCV, BVDV, and BDV isolates generically as pestiviruses. Nested amplification of the second region was designed to differentiate HCV from BVDV and BDV by exploiting relatively conserved differences in the nucleotide sequences that encode the major envelope glycoprotein. This second PCR correctly identified 36 of 36 diverse HCV isolates while failing to recognize any of 25 BVDV and BDV isolates. Multiple restriction fragment length analyses confirmed the identities of both external and nested PCR products. The two sets of PCRs may help confirm the generic identity of most pestiviruses and may permit presumptive differential diagnosis of HCV from BVDV and BDV.
The benomyl test as a fundamental diagnostic method for medical mycology
Authors: Summerbell R C
Published: January 2020

The fungicide benomyl has long been known to differentially affect major taxonomic groups of fungi. In the present study 163 species or aggregates of closely similar species of medically important fungi and actinomycetes, as well as species commonly isolated as clinical contaminants, were tested to determine their reactions to three concentrations of benomyl. Fungi of basidiomycetous, endomycetous, and microascaceous affinities were highly resistant, including all common yeasts and Geotrichum, Pseudallescheria, Scedosporium, and Scopulariopsis species. Also resistant were fungi of pleosporalean affinities with poroconidial anamorphs, such as Alternaria, Bipolaris, Curvularia, and Exserohilum species. Most other fungi of ascomycetous affinity were moderately to strongly susceptible. Such fungi included dermatophytes; Coccidioides, Blastomyces, and Histoplasma species; Sporothrix schenckii; medically important aspergilli; and "black yeasts." Benomyl testing aided in the provisional identification of nonsporulating mycelia, including common basidiomycetous isolates obtained as contaminants as well as nonsporulating Aspergillus fumigatus from pulmonary sources.
Restriction fragment length polymorphisms distinguish Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates from different geographical locations
Authors: Zuerner R L , Ellis W A , Bolin C A , Montgomery J M
Published: January 2020

Genetic variability among Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates representing several geographical regions was determined by restriction endonuclease analysis. Five previously unidentified EcoRI digestion patterns and one previously unidentified HhaI digestion pattern were seen with the various isolates. The copy number and genomic distribution of an L. borgpetersenii insertion sequence (IS1533) was determined. Hardjo-bovis isolate 033 (the type strain for hardjo-bovis) contained 40 well dispersed copies of IS1533. IS1533 probes were used to compare hardjo-bovis isolates by DNA blot hybridization analysis. Use of these probes showed the presence of additional genetic heterogeneity among hardjo-bovis isolates, which restriction endonuclease analysis did not show. Pulsed-field gel electrophoretic analysis of DNAs from several isolates suggested that some polymorphisms arose by genomic rearrangements. All hardjo-bovis isolates were categorized into 14 distinct groups on the basis of common hybridization and endonuclease digestion patterns. Most of these groups were isolated from distinct geographical regions, suggesting that several different clonal populations of hardjo-bovis exist.
Differentiation of mycobacteria on the basis of chemotype profiles by using matrix solid-phase dispersion and thin-layer chromatography
Authors: Hines M E , Frazier K S
Published: January 2020

Because of the rising incidence of clinical mycobacterial infections and the difficulty in identification and characterization of mycobacteria at the subspecies and serovar levels, a technique for differentiation that could be performed quickly and with relatively little equipment and expense was developed. Lysis and fractionation of mycobacteria by matrix solid-phase dispersion followed by thin-layer chromatography were used to produce chemotype profiles of the lipid and glycolipid components of each isolate. Organisms tested included Mycobacterium scrofulaceum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium flavescens, Mycobacterium kansasii, Mycobacterium bovis, 11 isolates of Mycobacterium gordonae, 10 serovars of Mycobacterium avium, and four strains of Mycobacterium paratuberculosis. A relative retention (Rx) value was established for each visible band on the chromatographs by comparison with a band common to all organisms tested. The chemotype profiles produced were different for every separate species and serovar sampled. These findings suggest that matrix solid-phase dispersion and subsequent thin-layer chromatography may have the sensitivity and flexibility to characterize and identify mycobacteria at the species and subspecies levels and potentially to identify clinical isolates of mycobacteria that have been difficult to identify by standard means. The technique has certain advantages over current biochemical, immunologic, and genetic methods because it is relatively simple to perform, is inexpensive, and requires a small amount of bacterial sample.
Analysis of the humoral response to the flagellin protein of Borrelia burgdorferi: cloning of regions capable of differentiating Lyme disease from syphilis
Authors: Robinson J M , Pilot-Matias T J , Pratt S D , Patel C B , Bevirt T S , Hunt J C
Published: January 2020

Selected regions of the Borrelia burgdorferi flagellin gene (fla) that exhibit high or low homology with related genes from other bacterial species were amplified by the polymerase chain reaction and expressed as fusion proteins in Escherichia coli. Purified fusion proteins were assayed for antibody reactivity in a microtiter plate enzyme-linked immunosorbent assay with sera from Lyme disease patients as well as syphilitic and normal sera. Immunoglobulin G antibody from Lyme disease patient sera reacted predominantly with the central portion of the protein. The region of the flagellin protein encompassing amino acids 64 to 311 detected nearly all of the immunoglobulin G-positive Lyme sera and only reacted with 1 of 26 syphilis patient serum samples. In contrast, 12 of 26 syphilis patient serum samples and 2 of 47 normal serum samples reacted with the amino terminus of the flagellin protein, whereas 4 of 26 syphilis patient serum samples and 7 of 47 normal serum samples reacted with the carboxyl terminus. The central region containing amino acids 64 to 311 may be employed diagnostically to differentiate antibodies to B. burgdorferi from antibodies to Treponema pallidum. In addition, this region also was recognized by immunoglobulin M in the Lyme patient sera, indicating its potential usefulness as a marker for early Lyme disease.
Possible role of specific immunoglobulin M antibodies to Plasmodium falciparum antigens in immunoprotection of humans living in a hyperendemic area, Burkina Faso
Authors: Boudin C , Chumpitazi B , Dziegiel M , Peyron F , Picot S , Hogh B , Ambroise-Thomas P
Published: January 2020

Two seroepidemiological studies were performed in an area of Burkina Faso hyperendemic for malaria to estimate the protective role of immunoglobulin M (IgM) antibodies. Six cross-sectional surveys were carried out on children (ages, < 16 years) in the village of Karankasso. The evolution of antibodies to crude extracts of Plasmodium falciparum (IgG or IgM antisomatic and IgG antiexoantigens) were tested by IFI or enzyme-linked immunosorbent assay and were followed up according to the fluctuations of the parasite densities. Specific IgG antibodies had the same evolution as parasite densities. By contrast, specific IgM antibodies increased when IgG and parasite densities began to decrease (despite a high inoculation rate). A longitudinal survey of 77 children and adults was conducted in another village (Dafinso). In that study, clinical follow-up of the selected individuals allowed us to define three groups in the population. Children in group 1 were considered nonimmune (children with one or more malaria attacks). Group 2 was composed of semiimmune children who did not present with any malarial attack during the survey but who had high levels of parasitemia during the transmission period. Group 3 was composed of immunoprotected adults. Specific IgM and IgG antibodies to crude extracts or a recombinant antigen (glutamate-rich protein) of P. falciparum were tested. Specific IgM antibodies were lower in group 1 (nonimmune) than in groups 2 (semiimmune) and 3 (immunoprotected). Furthermore, there was a negative correlation between parasite densities and the levels of specific IgM antibodies. We discuss the possible role of IgM antibodies in the acquisition of immunity to malaria.
Development of the polymerase chain reaction for diagnosis of chancroid
Authors: Chui L , Albritton W , Paster B , Maclean I , Marusyk R
Published: January 2020

The published nucleotide sequences of the 16S rRNA gene of Haemophilus ducreyi were used to develop primer sets and probes for the diagnosis of chancroid by polymerase chain reaction (PCR) DNA amplification. One set of broad specificity primers yielded a 303-bp PCR product from all bacteria tested. Two 16-base probes internal to this sequence were species specific for H. ducreyi when tested with 12 species of the families Pasteurellaceae and Enterobacteriaceae. The two probes in combination with the broad specificity primers were 100% sensitive with 51 strains of H. ducreyi isolated from six continents over a 15-year period. The direct detection of H. ducreyi from 100 clinical specimens by PCR showed a sensitivity of 83 to 98% and a specificity of 51 to 67%, depending on the number of amplification cycles.
Puumala virus antibody and immunoglobulin G avidity assays based on a recombinant nucleocapsid antigen
Authors: Kallio-Kokko H , Vapalahti O , Hedman K , Brummer-Korvenkontio M , Vaheri A
Published: January 2020

Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.
Five cases of primary amebic meningoencephalitis in Mexicali, Mexico: study of the isolates
Authors: Lares-Villa F , De Jonckheere J F , De Moura H , Rechi-Iruretagoyena A , Ferreira-Guerrero E , Fernandez-Quintanilla G , Ruiz-Matus C , Visvesvara G S
Published: January 2020

Five Naegleria strains isolated from patients with primary amebic meningoencephalitis and one strain isolated from the water of an artificial canal were investigated. All strains were pathogenic for mice when instilled intranasally and showed cytopathic effects in Vero cell cultures. Their growth characteristics (isolation and subculture at 45 degrees C), serological results, and isoenzyme patterns permitted us to identify the six strains as Naegleria fowleri. This is the first time that Naegleria fowleri has been isolated from patients with primary amebic meningoencephalitis in Mexico.
Biochemical characteristics and fatty acid composition of Gilardi rod group 1 bacteria
Authors: Moss C W , Daneshvar M I , Hollis D G
Published: January 2020

Fifteen strains of eugonic, nonoxidative, gram-negative rods isolated primarily from human wounds of the extremities and blood formed a distinct group which was designated Gilardi rod group 1. The phenotypic characteristics of Gilardi rod group 1 were most similar to those of CDC group M-5, with the major difference that nitrite reduction was observed with CDC group M-5. All 15 strains of Gilardi rod group 1 possessed a distinct fatty acid profile which was characterized by large amounts (> 15%) of cis-vaccenic (18:1 omega 7c), palmitic (16:0), myristic (14:0), and lactobacillic (19:0 cyc11,12) acids and moderate amounts (3 to 5%) of lauric (12:0), 3-hydroxylauric (3-OH-12:0), and palmitoleic (16:1 omega 7c) acids. This fatty acid profile is unique compared with the profiles of CDC group M-5 and other bacteria we have tested and is useful for the rapid identification of Gilardi rod group 1 isolates.
Culture confirmation of the carrier status of Babesia caballi-infected horses
Authors: Holman P J , Frerichs W M , Chieves L , Wagner G G
Published: January 2020

Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.
Use of the UV ParaLens adapter as an alternative to conventional fluorescence microscopy for detection of Pneumocystis carinii in direct immunofluorescent monoclonal antibody-stained pulmonary specimens
Authors: Mattia A R , Waldron M A , Sierra L S
Published: January 2020

The UV ParaLens light microscope adapter offers a useful and cost-effective alternative to conventional fluorescence microscopy for Pneumocystis carinii identification, particularly in AIDS patients. In a blinded study, in which 153 pulmonary specimens were examined for P. carinii by direct immunofluorescence, 40 of 42 specimens positive by fluorescence microscopy were also positive by ParaLens. No false positives were observed.
Gene encoding zonula occludens toxin (zot) does not occur independently from cholera enterotoxin genes (ctx) in Vibrio cholerae
Authors: Johnson J A , Morris J G , Kaper J B
Published: January 2020

Of 167 Vibrio cholerae isolates screened for sequences homologous with zonula occludens toxin (zot) or cholera toxin (ctx) genes, 3.0% of non-O1, 100.0% of clinical O1, and 0.0% of environmental O1 strains contained both zot and ctx. zot was present only in strains that were ctx positive; all ctx-positive strains carried zot. The absence of zot-positive, ctx-negative strains suggests ZOT is not an independent virulence factor for V. cholerae, although ZOT may play a role in the pathogenesis of toxigenic strains.
Effectiveness of resins in removing antibiotics from blood cultures
Authors: Crepin O , Roussel-Delvallez M , Martin G R , Courcol R J
Published: January 2020

The present study evaluates the effectiveness of BACTEC NR-26A resin-containing medium in removing eight antibiotics from blood cultures. Peak concentrations achievable clinically were chosen. All antibiotics were prepared in fresh human blood samples. Blood samples were obtained prior to and at 15 min and 2, 24, 48, and 72 h after inoculation. Aztreonam, ceftriaxone, imipenem, teicoplanin, and ticarcillin were partially removed from blood cultures.
Comparison of enterotoxin production, cytotoxin production, serogrouping, and antimicrobial susceptibilities of Clostridium difficile strains isolated from AIDS and human immunodeficiency virus-negative patients
Authors: Barbut F , Depitre C , Delmée M , Corthier G , Petit J C
Published: January 2020

We analyzed and compared Clostridium difficile strains isolated from diarrheic stools of 49 human immunodeficiency virus (HIV)-negative and 50 AIDS patients. Our results suggest that distribution patterns of serogroups are different in these two populations. Serogroup C (which has been previously reported to be very resistant to antimicrobial agents) represents 66.0 and 18.4% of the isolates from AIDS and HIV-negative patients, respectively (P < 0.001); the selection of serogroup C could be explained by multiple antibiotic pressure to which AIDS patients have been subjected.
UV red fluorescence of Eubacterium lentum
Authors: Mosca A , Strong C A , Finegold S M
Published: January 2020

Twenty-nine clinical isolates of Eubacterium lentum and two type species were evaluated for the ability to fluoresce under UV light. Twenty-one of the 29 isolates and both of the reference strains showed orange-to-red fluorescence. This fluorescence did not require blood or hemin in the culture media and did not fade upon air exposure. The fluorescent pigment, after extraction by 1 N NaOH, showed peak excitation at a wavelength of around 400 nm. The capacity of E. lentum to produce fluorescence may be a useful and time-sparing laboratory aid for its identification.
Cellular fatty acid compositions of "Achromobacter groups B and E"
Authors: Holmes B , Moss C W , Daneshvar M I
Published: January 2020

Strains of "Achromobacter groups B and E" were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. The CFA profiles of the two groups were identical and were distinct from CFA profiles of all other bacteria we have previously tested. These data provide support for results from whole-cell protein pattern analysis and DNA-DNA and rRNA-DNA hybridization studies, which show that "Achromobacter groups B and E" are biotypes of a single new genus and species.
Detection of Trichomonas vaginalis in pregnant women with the InPouch TV culture system
Authors: Draper D , Parker R , Patterson E , Jones W , Beutz M , French J , Borchardt K , McGregor J
Published: January 2020

Trichomonas vaginalis causes a common genitourinary infection which is frequently asymptomatic. At present, pregnant women are not usually screened for the infection unless they are symptomatic. In the present study, we screened and obtained samples for culture from all pregnant women attending a prenatal clinic with the InPouch TV culture system and compared results with those of standard culture in Diamond's medium and slide wet mount examination. The InPouch TV culture system was as reliable as Diamond's medium in detecting T. vaginalis and may be useful and effective in a pregnancy clinic setting.
Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction
Authors: Folgueira L , Delgado R , Palenque E , Noriega A R
Published: January 2020

We have evaluated the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculous infection. Two simple methods for mycobacterial DNA release have been compared: sonication and lysis with nonionic detergents and proteinase K. The more effective method was the enzymatic technique. By using this protocol with 75 specimens we detected M. tuberculosis DNA in all of the samples, whereas only 48 and 71 samples were positive by acid-fast staining and culture, respectively.
Characterization of unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles
Authors: Grattard F , Etienne J , Pozzetto B , Tardy F , Gaudin O G , Fleurette J
Published: January 2020

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.
Use of DNA analysis of Pasteurella haemolytica biotype T isolates to monitor transmission in bighorn sheep (Ovis canadensis canadensis)
Authors: Jaworski M D , Ward A C , Hunter D L , Wesley I V
Published: January 2020

Pneumonia has been identified as a major cause of poor lamb survival in indigenous herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in central Idaho. Pasteurella haemolytica was isolated from five adult Rocky Mountain bighorn ewes captured from a free-ranging herd in central Idaho. The lambs from two of these ewes delivered by cesarean section were free of P. haemolytica until 40 days of age and after repeated contact with their dams. The lambs subsequently developed signs of pneumonia, and P. haemolytica was isolated from nasal, pharyngeal, and transtracheal wash samples from each lamb. All P. haemolytica biotype T isolates from the ewes and lambs, as well as those from a 9-month-old lamb of the same herd from which samples for culture were obtained 2 years earlier, were subjected to HaeIII restriction enzyme analysis (REA) and ribotyping. Two ribotypes and seven REA patterns were visually distinguishable by these procedures. Similarity coefficients (SAB) of 0.09 to 0.95 were calculated for the seven REA patterns. The REA patterns of the isolates from the lambs were identical (SAB = 1.0). The isolates from the lambs also had SAB values of 1.0, which was indicative of identity with one of the seven isolates cultured from the ewes at the time of capture and with the organism isolated from the 9-month-old lamb. These procedures have the discriminatory capabilities necessary to monitor the transmission of specific strains of bacteria within and between animal populations.
Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East
Authors: Wolf M K , Taylor D N , Boedeker E C , Hyams K C , Maneval D R , Levine M M , Tamura K , Wilson R A , Echeverria P
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.
Controlled comparison of the BACTEC high-blood-volume fungal medium, BACTEC Plus 26 aerobic blood culture bottle, and 10-milliliter isolator blood culture system for detection of fungemia and bacteremia
Authors: Wilson M L , Davis T E , Mirrett S , Reynolds J , Fuller D , Allen S D , Flint K K , Koontz F , Reller L B
Published: January 2020

The BACTEC high-blood-volume fungal medium (HBV-FM) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) was compared with the Isolator (IS) tube and the BACTEC Plus 26 (BP26) blood culture bottle for the ability to recover fungi from the blood of adult patients suspected of having fungemia. A total of 6,836 blood culture sets that fulfilled criteria for inclusion in the study were received. Three separate comparisons were performed: 4,907 HBV-FM versus IS, 4,886 BP26 versus HBV-FM, and 4,949 BP26 versus IS. For the HBV-FM versus IS comparison, 218 isolates were recovered: 125 (57.3%) were bacteria and 93 (42.7%) were fungi. HBV-FM was comparable to IS for recovery of yeasts, but IS was superior for recovery of Histoplasma capsulatum (25 versus 0 isolates recovered [P < 0.001]). Growth of Torulopsis glabrata was detected earlier (P < 0.05) in HBV-FM bottles. For the BP26 versus HBV-FM comparison, 229 isolates were recovered: 161 (70.3%) were bacteria, and 68 (29.7%) were fungi. HBV-FM was superior for recovery of T. glabrata (P < 0.025) and all fungi combined (P < 0.025). There were no statistically significant differences in the speed of detection of microbial growth. For the BP26 versus IS comparison, 251 isolates were recovered: 165 (65.7%) were bacteria, and 86 (34.2%) were fungi. IS was superior for recovery of H. capsulatum (P < 0.001), T. glabrata (P < 0.05), and fungi other than H. capsulatum (P < 0.025). BP26 was superior for recovery of all bacteria combined (P < 0.001) and viridans group streptococci (P < 0.01). Growth of T. glabrata (P < 0.05) was detected earlier in IS tubes. Growth of Staphylococcus aureus (P < 0.01), viridans group streptococci (P < 0.01), Pseudomonas aeruginosa (P < 0.05), and all microorganisms combined (P < 0.05) was detected earlier in BP26 bottles. For yeast, 57 of 59 (96.6%), 79 of 80 (98.7%), and 64 of 67(95.5%) were recovered from BP26 bottles, HBV-FM bottles, and IS tubes, respectively, by day 14; for H. capsulatum, 14 of 36 (38%) isolates were recovered from IS tubes by day 14. Mean times of recovery were similar for BACTEC bottles and IS. We conclude that (i) for recovery of fungi from blood cultures, HBV-FM is equivalent to IS (with the exception of H. capsulatum); (ii) for recovery of bacteria, BP26 is superior to IS; (iii) BP26 bottles are inferior to both HBV-FM bottles and IS tubes for recovery of T. glabrata; and (iv) HBV-FM bottles must be paired with another blood culture bottle or system to optimize detection of bacteremia.
Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis
Authors: Daly J S , Worthington M G , Brenner D J , Moss C W , Hollis D G , Weyant R S , Steigerwalt A G , Weaver R E , Daneshvar M I , O'Connor S P
Published: January 2020

A Rochalimaea-like organism (strain F9251) was isolated from a patient with endocarditis after blood drawn for culture before antimicrobial therapy was subcultured onto blood and chocolate agars and incubated for 2 weeks in 5% CO2. The strain was phenotypically similar to known Rochalimaea species. The cellular fatty acid composition of strain F9251 was close to but distinct from those of the three known Rochalimaea species and was most similar to that of R. vinsonii. Labeled DNA from strain F9251 was 59 to 67% related to DNAs from type strains of the three described Rochalimaea species, and its 16S rRNA gene sequence was 98.9% or more homologous to their 16S rRNA gene sequences. These findings support classification of F9251 as a new Rochalimaea species, for which the name Rochalimaea elizabethae sp. nov. is proposed. The patient infected with the organism had large bacterial vegetations on his aortic valve and was cured with antibiotics and valve-replacement surgery. Recognition of the procedures required to identify this and other Rochalimaea species suggests that clinical laboratories should prolong the incubation times of cultures of blood and tissue from patients with suspected endocarditis, patients with fever of unknown origin, and immunocompromised patients with fever so that the full spectrum of disease caused by these organisms can be recognized.
Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay
Authors: Young K K , Resnick R M , Myers T W
Published: January 2020

Amplification of RNA by the polymerase chain reaction (PCR) is normally a two-step process requiring separate enzymes and buffer conditions. We describe a combined reverse transcription-PCR (RT-PCR) assay for hepatitis C virus (HCV) RNA amplification in which a single enzyme and buffer condition are used. In this assay, both the RT and PCR steps are carried out with the thermoactive DNA polymerase of Thermus thermophilus. A transcription vector containing HCV sequences has also been constructed to generate quantifiable HCV RNA templates that can be used to optimize reaction conditions and to assess the efficiency of amplification. Amplification from < or = 100 copies of RNA was detected reproducibly by gel electrophoresis. The assay sensitivity was increased to 10 RNA copies by hybridization to a probe. The patterns of viremia in three individuals infected with HCV were examined by amplification of HCV RNA from plasma samples collected serially over a period of 1 year. These results were correlated with the times of seroconversion and the onset of rise in levels of alanine aminotransferase in serum. In all three subjects, HCV RNA was detected prior to seroconversion and the initial rise in levels of alanine aminotransferase in serum. Upon seroconversion, HCV RNA fell to a level below the detection limit of the assay. This pattern of transient viremia appears to be characteristic of acute, resolving HCV infections. The combined RT-PCR assay is a sensitive method which circumvents the problems associated with PCR amplification of RNA. Using this assay, we demonstrated that three donors infected by the same index case all have similar patterns of viremia.
Determination of carbohydrate profiles of Bacillus anthracis and Bacillus cereus including identification of O-methyl methylpentoses by using gas chromatography-mass spectrometry
Authors: Fox A , Black G E , Fox K , Rostovtseva S
Published: January 2020

Bacillus anthracis and Bacillus cereus are closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically. It is well known that vegetative and spore forms of bacilli are quite distinct both morphologically and chemically, but spore-specific chemical markers allowing these species to be distinguished have not been previously described. By using gas chromatography-mass spectrometry, vegetative cells and spores of the two species were shown to exhibit distinct carbohydrate profiles. Profiles of vegetative B. anthracis typically contained high levels of galactose but did not contain galactosamine, whereas B. cereus contained galactosamine and generally low levels of galactose. Spore cultures exhibited unique carbohydrate profiles compared with those of vegetative cultures. B. anthracis spore profiles contained rhamnose alone, whereas B. cereus spore profiles contained rhamnose and fucose. Additionally, two spore-specific O-methylated methylpentoses were discovered. Both B. anthracis and B. cereus spores contained 3-O-methyl rhamnose, whereas B. cereus spores also contained 2-O-methyl rhamnose. Carbohydrate profiling is demonstrated to be a powerful tool for differentiating the two closely related species. Differentiation does not depend on whether organisms are in the vegetative or spore stage of growth.
Evaluation of polymerase chain reaction amplification of Mycobacterium leprae-specific repetitive sequence in biopsy specimens from leprosy patients
Authors: Yoon K H , Cho S N , Lee M K , Abalos R M , Cellona R V , Fajardo T T , Guido L S , Dela Cruz E C , Walsh G P , Kim J D
Published: January 2020

Biopsy specimens were obtained from 102 leprosy patients before chemotherapy and examined by polymerase chain reaction (PCR) using the primers amplifying the 372-bp DNA of a repetitive sequence of Mycobacterium leprae. The PCR results were then compared with bacterial indices (BI) of slit-skin smears and biopsy specimens. The intensities of DNA bands were in general correlated with the numbers of acid-fast bacilli, and even a sample with only one organism gave a PCR positive result. Ten 5-micron sections from each frozen tissue sample were pooled and processed for DNA preparation. PCR was positive for 11 (73.3%) of 15 biopsy specimens with BI of 0 determined for the paraffin sections from the same biopsy samples. PCR also gave positive results for 84 (96.6%) of 87 BI positive biopsy samples. Although the difference in overall results between the two methods was not statistically significant, PCR seemed to have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli. Further evaluation of PCR using more specimens from leprosy patients who are bacteriologically negative is warranted to ensure PCR's advantage over the conventional microscopic examination for the diagnosis of leprosy.
Evaluation of commercial antisera for serotyping heat-labile antigens of Campylobacter jejuni and Campylobacter coli
Authors: Nicholson M A , Patton C M
Published: January 2020

Commercial antisera for serotyping 22 heat-labile antigens of Campylobacter jejuni and Campylobacter coli were evaluated by using 66 isolates from human and nonhuman sources. Test results were compared with results of tests using antisera produced at the Centers for Disease Control (CDC), Atlanta, Ga. All strains (three isolates of each of the 22 serotypes) were typeable with the CDC antisera. Of 66 test strains, 39 (59%) were typed as the same serotype with both sets of antisera. Twenty-four strains (36%), including two heat-labile serotype reference strains, were nonreactive with the commercial antisera, and three strains (4.5%) were typed as serotypes different from those obtained with CDC antisera. Five of the 22 commercial antisera correctly serotyped all homologous strains. Our study indicated that two polyvalent antiserum pools, 7 unabsorbed antisera, and 16 absorbed monovalent antisera are weak and need modification to enhance their antibody titers. Further studies are necessary to explain the antigenic change to a different serotype in three strains.
Identification of group-common linear epitopes in structural and nonstructural proteins of enteroviruses by using synthetic peptides
Authors: Cello J , Samuelson A , Stålhandske P , Svennerholm B , Jeansson S , Forsgren M
Published: January 2020

Synthetic peptides were employed in enzyme-linked immunosorbent assays to identify group-common linear epitopes in the structural and nonstructural proteins of enteroviruses. Nine linear epitopes were recognized by using sera from patients with heterotypic immunoglobulin G antibody responses to enterovirus infections. The most-reactive peptides were derived from conserved regions of the amino-terminal part of VP1, whereas peptides representing sequences from other conserved regions of VP1, as well as VP2, VP3, and VP4, and from a nonstructural region showed no or poor reactivity. These findings may be useful in the development of serological tests for the diagnosis of infections caused by a broad range of enteroviruses.
Competitive radioimmunoassay to detect antibodies to herpes B virus and SA8 virus
Authors: Norcott J P , Brown D W
Published: January 2020

A monoclonal competitive radioimmunoassay (CompRIAm) which detects antibody to herpesvirus simiae (B virus) in monkey and human sera and antibody to SA8 virus in monkey sera but not antibody to herpes simplex virus in human sera is described. Of 232 serum samples from wild-caught cynomolgus monkeys, 117 serum samples were positive when tested by CompRIAm. The results were in close agreement (97.5%) with B virus neutralizing antibody results on the same sera. Sera from 97 wild-caught rhesus monkeys and 92 wild-caught baboons were also tested. The CompRIAm was able to differentiate between sera that had neutralizing antibody to B virus and SA8 virus and those that did not, although the discrimination was not as clear as that in the tests on cynomolgus monkey sera. Sequential sera from two humans with confirmed cases of B virus infection were tested by CompRIAm. B virus antibody was detected in sera from both humans. None of 237 other serum samples from blood donors and patients attending sexually transmitted disease clinics reacted in the CompRIAm.
Identification of Bacteroides forsythus in subgingival plaque from patients with advanced periodontitis
Authors: Gersdorf H , Meissner A , Pelz K , Krekeler G , Göbel U B
Published: January 2020

Bacteroides forsythus has been associated with destructive adult periodontitis. Up to now, detailed analysis by classical means was hampered by the fastidious nature of the organism. There is hope that the application of molecular detection methods such as indirect immunofluorescence or in situ hybridization (ISH) will allow for more rapid and accurate identification. Here we describe a B. forsythus-specific probe (BFV530), complementary to 16S rRNA, which correctly identified all B. forsythus isolates as confirmed by biochemical, protein, or fatty acid analysis. To assess whether this probe might be suitable for direct identification of B. forsythus in clinical specimens, a total of 92 subgingival plaque samples were analyzed. Fifty-five specimens were tested in parallel by culture, light microscopy, and filter hybridization. Unfortunately, the overall agreement between results of filter hybridization and conventional methods was 70.9% only. We therefore examined 37 new specimens by ISH and indirect immunofluorescence by using fluorescently labeled probe BFV530 or B. forsythus-specific monoclonal antibody 116BF1.2 (kindly provided by R. Gmür, Zurich, Switzerland), respectively. Agreement between these methods was 100%, indicating that ISH with probe BFV530 might be used to accurately identify B. forsythus directly in subgingival plaque samples.
Comparison of the Syva MicroTrak enzyme immunoassay and Gen-Probe PACE 2 with cell culture for diagnosis of cervical Chlamydia trachomatis infection in a high-prevalence female population
Authors: Clarke L M , Sierra M F , Daidone B J , Lopez N , Covino J M , McCormack W M
Published: January 2020

Culture is currently considered the "gold standard" for detecting Chlamydia trachomatis infections. We evaluated the Syva MicroTrak enzyme immunoassay (EIA) and Gen-Probe PACE 2 tests, which detect chlamydial antigens and rRNA, respectively. These assays were compared with each other and with culture for the detection of C. trachomatis in cervical specimens obtained from 217 women attending a clinic for sexually transmitted diseases. The prevalence of infection was 22.1% by culture. The sensitivity, specificity, and positive and negative predictive values were 79.2, 98.2, 92.6, and 94.3%, respectively, for EIA. For PACE 2, the respective values were 77.1, 97.6, 90.1, and 93.7%. After corrections for two false-negative cultures, the sensitivities and specificities were 80 and 99.4%, respectively, for the EIA and 78 and 98.8%, respectively, for the probe assay. Quantitative evaluation of the results showed that false-negative results with either assay were associated with cultures that had low inclusion counts or were negative without subpassage. Analysis of nonculture results revealed that 2.3% of the EIA results and 4.6% of the probe assay results were within +/- 30% of the respective assay cutoff values. These included four false-negative (one EIA and three probe) and two false-positive (one EIA and one probe) results. The Syva MicroTrak EIA and the Gen-Probe PACE 2 assay are comparable to but significantly less sensitive than culture. Use of a grey zone may help identify the need for repeat or confirmatory testing.
Rapid screening test for enterotoxin-producing Bacillus cereus
Authors: Jackson S G
Published: January 2020

Culture supernatants of 30 enterotoxin-producing Bacillus cereus isolates produced a characteristic progressive destruction of McCoy cell monolayers. Enterotoxin-negative B. cereus and other group 1 Bacillus spp. caused no monolayer disruption. The McCoy cell tissue culture system appears to provide a rapid screening assay for detection of enterotoxin-producing B. cereus.
Random amplified polymorphic DNA assay is less discriminant than pulsed-field gel electrophoresis for typing strains of methicillin-resistant Staphylococcus aureus
Authors: Saulnier P , Bourneix C , Prévost G , Andremont A
Published: January 2020

Twenty-six strains of methicillin-resistant Staphylococcus aureus with different pulsed-field gel electrophoresis fingerprints were tested by random amplified polymorphic DNA assay with three primers, resulting in 15 to 20 different random amplified polymorphic DNA fingerprints. By summing the results for the three primers, the number of different fingerprints increased to 25, but two strains could not be differentiated. We conclude that pulsed-field gel electrophoresis remains the best method of typing methicillin-resistant S. aureus strains.
Detection of serum antibodies to Chlamydia trachomatis in patients with chlamydial and nonchlamydial pelvic inflammatory disease by the IPAzyme Chlamydia and enzyme immunoassay
Authors: Mattila A , Miettinen A , Heinonen P K , Teisala K , Punnonen R , Paavonen J
Published: January 2020

A novel serological test, IPAzyme Chlamydia (Savyon Diagnostics Ltd., Beer Sheva, Israel), was compared with an enzyme immunoassay (EIA) for the ability to detect serum immunoglobulin G and A antibodies in the diagnosis of acute chlamydial pelvic inflammatory disease. In comparison with cell culture, which is the "gold standard," IPAzyme Chlamydia and EIA exhibited sensitivities of 63 and 68% and specificities of 76 and 87%, respectively. Thus, IPAzyme Chlamydia offers no advantages over the EIA, and neither serological test can be recommended for the diagnosis of acute Chlamydia trachomatis infection. So far, conventional cell culture remains the most reliable diagnostic test for chlamydial pelvic inflammatory disease.