bluuebunny/crossref_metadata_2025 huggingface dataset:
Class-specific immune response to Yersinia enterocolitica serotype O9 antigens as determined by enzyme-linked immunosorbent assay
Authors: Fernández-Lago L , Santoyo F J , Vizcaíno N , Chordi A
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide (S-LPS) as the antigen was used to analyze the antibody response in rabbits orogastrically and intravenously infected with virulent (plasmid-bearing) Yersinia enterocolitica O9 strains (pYV+) and with the avirulent (plasmid-cured) derivatives (pYV-). A significative response of immunoglobulin G (IgG), IgA, and IgM antibodies against the S-LPS antigen was evident in sera from the rabbits orogastrically infected with pYV+ strains. This immune response was stronger and persisted longer than those obtained with the corresponding pYV- strains. In contrast, few differences were observed in the titers and evolution of IgG, IgA, and IgM antibodies against the S-LPS antigen in rabbits intravenously infected with pYV+ and pYV- strains. These results suggest that the necessity of the virulence plasmid for the establishment of infection by Y. enterocolitica serotype O9 is conditioned by the infection route used. When the S-LPS ELISA was compared with the radial immunodiffusion test using the native hapten as the antigen, the results showed that the ELISA technique was more sensitive. However, only those sera obtained between 2 and 8 weeks postinfection from rabbits intravenously infected with plasmid-bearing strains were positive in the radial immunodiffusion test.
Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies
Authors: Wang S P , Grayston J T
Published: January 2020

A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described. Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed. The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories. It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test. Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous.
Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis
Authors: Suchland R J , Stamm W E
Published: January 2020

Serotyping of Chlamydia trachomatis strains usually requires three to six serial passages in shell vials to attain sufficient antigen for typing procedures. To circumvent this problem, we developed a rapid low-passage method for serotyping of C. trachomatis clinical isolates. Isolates with an inclusion count of greater than or equal to 500 per well in primary isolation were inoculated directly onto cell culture monolayers in microtiter plates for typing. Primary isolates with a lower initial inclusion count were passed one to two times in shell vials until there were greater than or equal to 20 inclusions per well and were then inoculated onto plates for typing. Inclusions were grown to maturity and reacted with a panel of 17 C. trachomatis-specific monoclonal antibodies in pools. Wells were then reacted with a fluorescein isothiocyanate conjugate and read by FA microscopy, and the reaction patterns were compared with prototype strain reaction patterns to determine the serotype. By the microtiter method, we successfully typed 1,711 consecutive C. trachomatis isolates; 1,215 isolates (71%) were typed with no or with one passage. The first 209 isolates typed by the microtiter method were also typed by the dot-enzyme-linked immunosorbent assay serotyping method; 100% agreement was demonstrated among strains that were typeable by both methods. We conclude that the microtiter method is extremely useful for accurate serotyping of large numbers of isolates and requires greatly reduced technician time.
Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis
Authors: Tyler S D , Johnson W M , Lior H , Wang G , Rozee K R
Published: January 2020

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.
DNA restriction fragment length polymorphism differentiates crossed from independent infections in nosocomial Xanthomonas maltophilia bacteremia
Authors: Bingen E H , Denamur E , Lambert-Zechovsky N Y , Bourdois A , Mariani-Kurkdjian P , Cezard J P , Navarro J , Elion J
Published: January 2020

Restriction fragment length polymorphisms of total DNA and rDNA were used to study the relationship between 11 isolates of Xanthomonas maltophilia, obtained from seven patients with nosocomial bacteremia in four distinct wards of a single hospital, and the type strain of the species, ATCC 13637. Our results indicated that there were episodes of cross-infection among the patients of two wards, but there were also independent infectious episodes in the two other wards.
Monoclonal antibodies specific for the phase-variant O-acetylated K1 capsule of Escherichia coli
Authors: Torensma R , van Wijk A , Visser M J , Bouter A , Rozenberg-Arska M , Verhoef J
Published: January 2020

Two monoclonal antibodies, one each of the immunoglobulin M and G2b types, were produced from mouse spleen cells. These monoclonal antibodies only reacted with approximately 50% of the Escherichia coli K1 strains and not against group B meningococci. No reaction was observed after the strains were boiled. E. coli K1 strains that reacted with the monoclonal antibodies could become nonreactive after subculture. Based on these findings, we conclude that the monoclonal antibodies react with the O-acetylated K1 capsule.
Rotavirus-associated diarrhea in rural Bangladesh: two-year study of incidence and serotype distribution
Authors: Fun B N , Unicomb L , Rahim Z , Banu N N , Podder G , Clemens J , Van Loon F P , Rao M R , Malek A , Tzipori S
Published: January 2020

Stools were evaluated from 5,811 patient visits for treatment of diarrhea in Matlab, Bangladesh, between June 1987 and May 1989. The stools were analyzed to determine the distribution of serotypes of group A rotaviruses (RV). A total of 898 stool samples (15.5%) contained RV, as determined by using an enzyme-linked immunosorbent assay. RV isolates from 855 of these samples were serotyped by using serotype-specific synthetic oligonucleotide probes. A total of 558 (65.3%) could be assigned to specific serotypes: 166 (19.4%), 228 (26.7%), 39 (4.6%), and 125 (14.6%) belonged to serotypes 1 through 4, respectively; 12 (1.4%) hybridized with more than one serotype; and 285 (33.3%) failed to hybridize. RV diarrhea was evident throughout the year, with peaks in the dry winter months and in September 1988, coinciding with a major flood. RV was isolated from 46.6% of patients between 7 and 12 months old. Among children under 24 months of age with RV diarrhea, 1.2% (10 of 828) died. The corresponding percentage for children with diarrhea from all causes is 0.9% (29 of 3,301).
Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains
Authors: Pearce M A , Howell S A
Published: January 2020

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, BglII, and HinfI on a collection of Candida albicans strains comprising eight strains randomly selected from clinical microbiology laboratory specimens, three reported azole-resistant strains from treatment failures, and several subcultures of the azole-resistant strain NCPF 3310 (also known as the Darlington strain) received from different laboratories. The results demonstrated a diversity of the restriction fragment length polymorphism patterns that were obtained and revealed that two of the proposed Darlington subcultures had patterns distinct from each other and from those of the other Darlington isolates; both were also found to have lost their azole resistance.
Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures
Authors: Verstijnen C P , Ly H M , Polman K , Richter C , Smits S P , Maselle S Y , Peerbooms P , Rienthong D , Montreewasuwat N , Koanjanart S
Published: January 2020

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.
Protection of agammaglobulinemic piglets from porcine rotavirus infection by antibody against simian rotavirus SA-11
Authors: Lecce J G , Leary H L , Clarke D A , Batema R P
Published: January 2020

Rotavirus, a double-stranded RNA virus, has been implicated as a diarrhea-provoking agent in a variety of animal species. Several previous reports have shown that immunization with a single serotype may result in increased in vitro neutralization titers against serotypes not represented in the immunogen. This study was undertaken to determine whether antibody from cows immunized against simian rotavirus strain SA-11 (which is alien to pigs) could protect neonatal piglets from infection with a North Carolina isolate of porcine rotavirus. Accordingly, cows were immunized with SA-11 and an immunoglobulin G (IgG)-rich fraction was isolated from their colostrum. An IgG-rich fraction was similarly isolated from colostrum of nonimmunized cows. At equal concentrations, IgG from SA-11-immunized cows had two- to fourfold higher neutralization titers to seven of eight test strains of rotavirus, including SA-11 (serotype 3); human rotavirus serotypes 1, 3, and 4; North Carolina porcine rotavirus (serotype undetermined); Ohio State porcine rotavirus (serotype 5); and bovine rotavirus (serotype 6). The IgG-rich fractions were fed as dietary supplements to agammaglobulinemic piglets infected with the North Carolina porcine rotavirus. IgG from the SA-11-immunized cows was about eightfold more effective in protecting piglets than was IgG from nonimmunized cows.
Fine serotyping of human immunodeficiency virus serotype 1 (HIV-1) and HIV-2 infections by using synthetic oligopeptides representing an immunodominant domain of HIV-1 and HIV-2/simian immunodeficiency virus
Authors: Baillou A , Janvier B , Leonard G , Denis F , Goudeau A , Barin F
Published: January 2020

In this study, enzyme immunoassays for detection of type-specific antibodies to human immunodeficiency viruses (HIV) were developed by using short peptides corresponding to sequences located within the immunodominant domain of the transmembrane glycoproteins of both HIV-1 and HIV-2-simian immunodeficiency virus (SIV). The assays were highly sensitive with currently available sera from various geographical areas. Furthermore, they appeared to be more specific in HIV serotyping than the Western blot (immunoblot) assay, since all of the sera were clearly discriminated as one or the other type. It was also shown that in contrast to HIV-1, the C-terminal cysteine residue (amino acid 620, SIV from captive macaques, Mm142 strain) of the HIV-2-SIV peptide is not necessary for recognition of the peptide by antibody to HIV-2.
Immunoblot analysis of the humoral immune response to Leishmania donovani infantum polypeptides in human visceral leishmaniasis
Authors: Rolland-Burger L , Rolland X , Grieve C W , Monjour L
Published: January 2020

Using the immunoblot technique, we have compared the reactions of Leishmania donovani infantum polypeptides with the immunoglobulin G of human sera from patients with parasitologically proven L. d. infantum infection, with suspected visceral leishmaniasis, and with other leishmaniases, protozoiases, helminthiases, and fungal or bacterial diseases. A 94-kDa component reacted with all L. d. infantum-infected sera and with 75% of sera from patients with clinical and serological but no parasitological diagnoses. No reaction was observed with sera from patients in the other disease groups or with control sera. Studies of eight different isolates, subspecies, and species of the genus Leishmania demonstrated that the 94-kDa component was expressed in all strains examined.
Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents
Authors: Nato F , Mazie J C , Fournier J M , Slizewicz B , Sagot N , Guibourdenche M , Postic D , Riou J Y
Published: January 2020

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents.
Localization of endogenous activity of phospholipases A and C in Ureaplasma urealyticum
Authors: De Silva N S , Quinn P A
Published: January 2020

Endogenous activities of phospholipases A and C in Ureaplasma urealyticum were assayed in cellular fractions of exponential-phase cells. Enzymatic studies indicated that ATPase activity was localized in the plasma membrane fraction and NADH and NADPH dehydrogenase activities were localized in the cytosol fraction. Studies with purified ureaplasma membranes demonstrated that, of three serovars tested, endogenous phospholipase A1, A2, and C activities were localized in the plasma membrane. Very low levels of activity were observed in the cytosol fractions. Phospholipase A2 activity in the plasma membrane was 3- to 5-fold higher than the activity in the lysates and 60- to 300-fold higher than the activity of phospholipase A1. Phospholipase C was localized mainly in the plasma membrane, with 20% found in the cytosol fraction. The levels of activity were comparable among the three serovars. There was a significantly lower level of activity in cells from the stationary growth phase than in the exponential phase. Significant differences were observed in the phospholipase A activities among the U. urealyticum serovars 3, 4, and 8. Phospholipase A2 activity was twofold higher in serovar 8 membranes, and phospholipase A1 activity was twofold higher in serovar 3 membranes. These results demonstrate that endogenous activities of phospholipase A and C are localized primarily in the plasma membrane fraction of U. urealyticum. The specific activities in the membranes of the phospholipases varied among the three serovars. Phospholipase enzymes may function as virulence factors in U. urealyticum and may vary among the serovars.
Optimization of a rapid nonisotopic DNA probe assay for Plasmodium falciparum in the Gambia
Authors: McLaughlin G L , Decrind C , Dayal-Drager R , Hassan-King M , Subramanian S , Greenwood B M
Published: January 2020

An enzyme-linked synthetic DNA probe which hybridizes to repetitive DNA of Plasmodium falciparum was used in conjunction with a microtiter-based lysis and filtration blood processing procedure. An assay protocol was developed that is more sensitive and robust than previous protocols, which use stored blood and phenol extraction. In comparison with thick smear examination, 33% positive, 60% negative, and 7% conflicting scores were recorded from 390 analyzed clinical samples, and the sensitivity threshold was about 30 parasites per mm3 of blood.
Spotted fever group rickettsiae or Borrelia burgdorferi in Ixodes cookei (Ixodidae) in Connecticut
Authors: Magnarelli L A , Swihart R K
Published: January 2020

Immatures and females of Ixodes cookei, a hard-bodied tick, were collected from woodchucks and other mammals in the northeastern United States and examined for spotted fever group rickettsiae and Borrelia burgdorferi. Of the 93 nymphs analyzed by a hemolymph test, 4 (4.3%) harbored rickettsiae. Six (15%) of 40 females were also infected. All infected ticks were collected from woodchucks in Connecticut. Indirect fluorescent antibody staining of midgut tissues from 128 nymphs revealed B. burgdorferi in two (1.6%) ticks, whereas larval and female ticks were negative. Further consideration should be given to I. cookei as a possible vector of spotted fever group rickettsiae or spirochetes that cause Lyme borreliosis.
Measurement of antibodies to varicella-zoster virus by using a latex agglutination test
Authors: Steinberg S P , Gershon A A
Published: January 2020

We evaluated a rapid, simple-to-perform assay for detecting antibodies to varicella-zoster virus by latex agglutination (LA); dilutions of test sera were added to latex particles coated with varicella-zoster virus antigen. We tested 878 serum specimens by LA and with fluorescent antibody to membrane antigen; of these, 227 were also tested by a commercially marketed enzyme-linked immunosorbent assay (ELISA). LA was almost as sensitive as the fluorescent antibody to membrane antigen test and more sensitive than ELISA. No cross-reacting antibodies were detected by LA, and false-positive reactions were rare.
Evaluation of the rapid CLEARVIEW Chlamydia test for direct detection of chlamydiae from cervical specimens
Authors: Stratton N J , Hirsch L , Harris F , de la Maza L M , Peterson E M
Published: January 2020

The CLEARVIEW Chlamydia test (Unipath, Mountain View, Calif.), a 30-min immunoassay, was compared to a standard tissue culture technique for the direct detection of chlamydiae from 677 cervical specimens obtained from 667 patients. For data analysis, 15 specimens were eliminated because of toxicity in the culture and 14 were eliminated because of failure of the extracted specimen to migrate in the CLEARVIEW test, one of the latter group being culture positive. Of the remaining 648 specimens, 40 were culture positive, of which 38 were detected by the CLEARVIEW test, and 12 specimens were positive only by the CLEARVIEW test. Therefore, the CLEARVIEW in comparison with culture was easier to perform, more rapid, and in this low prevalence (6.2%) population had a 95.0% sensitivity, 98.0% specificity, and 76.0% positive and 99.7% negative predictive values.
Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome
Authors: Hauser A R , Stevens D L , Kaplan E L , Schlievert P M
Published: January 2020

Toxic shock-like syndrome (TSLS) is characterized by hypotension or shock, fever, multiorgan system involvement, and a concurrent group A streptococcal infection. We analyzed 34 streptococcal strains isolated from patients with clinically well-documented TSLS for their pyrogenic toxin profiles and M-protein types. Although strains of nine different M types were represented in the sample, 74% of the isolates were of either M type 1 or 3. It was determined that 53% produced streptococcal pyrogenic exotoxin type A under in vitro growth conditions and that 85% contained the gene encoding this toxin. These values are in contrast to the published value of 15% for the incidence of this gene in a sample of general group A streptococcal isolates. As has been found with all group A streptococci examined to date, regardless of disease association, 100% of TSLS-associated isolates contained the gene encoding pyrogenic exotoxin type B. This toxin was detectably produced by 59% of isolates. The gene encoding pyrogenic toxin type C was found in only 21% of isolates. We conclude that the pyrogenic exotoxin type A gene is associated with group A streptococcal strains isolated from patients with TSLS and may play a causative role in this illness. However, other factors are also likely to be important, since not all strains from patients with TSLS contained the A toxin gene.
Disk diffusion method for susceptibility testing of Neisseria gonorrhoeae
Authors: Ringertz S , Rylander M , Kronvall G
Published: January 2020

The standard medium for disk diffusion and MIC testing of Neisseria gonorrhoeae (that of the National Committee for Clinical Laboratory Standards) was tested to establish zone correlations for the MIC breakpoints currently used in Sweden. Eight gonococcal control strains representing both susceptible and resistant strains and 50 clinical isolates were tested. The standard medium did not support the growth of two control strains and three clinical isolates when the standardized inoculum was used in the disk diffusion test. The same medium with the addition of hemoglobin was introduced. This medium supported the growth of all strains. The correlations between the MICs and the zones of inhibition were calculated for penicillin, ampicillin, cefuroxime, erythromycin, tetracycline, doxycycline, ciprofloxacin, and spectinomycin. The range of MICs for the clinical isolates were broad, without bimodal distribution, for all antibiotics, except ciprofloxacin and spectinomycin. With the susceptibility distribution of MICs and zones near the current susceptible and intermediate or intermediate and resistant limits, a low reproducibility of tests and a high frequency of minor interpretive errors can be expected. A revision of MIC breakpoints seems warranted but can only be done after renewed clinical evaluation of different treatment regimens.
Characterization of an immunoreactive species-specific 19-kilodalton outer membrane protein from Helicobacter pylori by using a monoclonal antibody
Authors: Drouet E B , Denoyel G A , Boude M , Wallano E , Andujar M , de Montclos H P
Published: January 2020

Immunoblotting experiments on hyperimmune rabbit serum and sera from patients with Helicobacter pylori gastritis showed a consistent antibody response to a 19-kDa outer membrane protein antigen. A monoclonal antibody, designated HP 40, which reacted by Western immunoblotting with this protein was produced. It was shared by all H. pylori strains tested (D 273, NCTC 11637, and 24 wild strains) but not by the thermophilic Campylobacter species, Campylobacter fetus, Helicobacter mustellae, or Helicobacter fennelliae. Immunogold staining suggested that the 19-kDa antigen was exposed on the outer surface of the bacteria. Its functional role and effectiveness as a serological diagnostic tool are under study.
Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples
Authors: Ball H J , Finlay D , Mackie D P , Greer D , Pollock D , McNair J
Published: January 2020

A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs.
Rapid clearance of Candida albicans mannan antigens by liver and spleen in contrast to prolonged circulation of Cryptococcus neoformans antigens
Authors: Kappe R , Müller J
Published: January 2020

Clearances of mannan antigen from Candida albicans and glucuronoxylomannan antigen from Cryptococcus neoformans were examined in nonimmune rabbits by using self-prepared latex agglutination tests. Injected intravenously, 20 mg of Candida mannan antigen was cleared from the serum with a half-life of approximately 2 h. In contrast, 20 micrograms of Cryptococcus glucuronoxylomannan antigen had a half-life in serum of approximately 24 h. At the latest, 9 h after injection, both antigens were no longer detectable without pretreatment of serum samples with protease and heating to 100 degrees C, thus indicating rapid binding by serum proteins other than immunoglobulins. Candida mannan antigen clearance was also examined in nonimmune mice after intravenous injection of (i) 200 micrograms of Candida mannan antigen, which accumulated in the liver and spleen and persisted for 97 days; (ii) 2 x 10(7) ethanol-killed Candida blastospores, which was accompanied by rapid clearance of mannan from the blood but accumulation of mannan in the liver and spleen and slow clearance from these organs; (iii) 6 x 10(6) viable C. albicans cells (lethal infection), which resulted in a rapid decrease of Candida CFU in the blood, liver, and spleen during the first 8 h, after which blood cultures were negative on day 2 and viable Candida burdens in the liver and spleen persisted at 10(5) CFU/g, whereas Candida mannan antigen continued to circulate in the bloodstream and accumulated in the liver and spleen.
Distinction of deep versus superficial clinical and nonclinical isolates of Trichosporon beigelii by isoenzymes and restriction fragment length polymorphisms of rDNA generated by polymerase chain reaction
Authors: Kemker B J , Lehmann P F , Lee J W , Walsh T J
Published: January 2020

Fifteen clinical and environmental strains of Trichosporon beigelii were analyzed for similarities by using morphological features, biochemical profiles based on carbon compound assimilation and uric acid utilization, isoenzyme electrophoresis, and restriction fragment length polymorphisms of a segment of genes coding for rRNA expanded with the polymerase chain reaction. The findings suggest that strains that cause invasive disease are distinct from the superficial and the nonclinical isolates and that isolates from the skin and mucosae represent a number of different organisms, including some environmental forms. The study shows that T. beigelii is a complex of genetically distinct organisms and that more than one type is found in clinical samples.
Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody
Authors: Martin S J , Siebeling R J
Published: January 2020

A serotyping scheme for Vibrio vulnificus predicated on the detection of lipopolysaccharide (LPS) antigens is proposed. The serovar O typing scheme used to type V. vulnificus employs polyclonal antisera raised in rabbits immunized with heat-killed whole-cell vaccines. Polyclonal typing sera produced in this manner cross-react with heterologous strains. Affinity purification of polyclonal antisera with LPS affinity columns resolved some of these cross-reactions; however, affinity-purified polyclonal antisera still showed cross-reactions that were nonreciprocal. On the basis of the serological patterns that were obtained with affinity-purified polyclonal antisera, V. vulnificus strains were selected as vaccine strains for production of monoclonal antibody. Spleen cells harvested from BALB/c mice immunized with formalin-killed V. vulnificus cells were fused with SP2/O-Ag 14 myeloma cells. Hybridomas were screened by using LPS and whole-cell enzyme-linked immunosorbent assay to identify clones secreting LPS-specific antibodies. Monoclonal antibodies identified five LPS serological varieties of V. vulnificus and a single serovar each for Vibrio damsela and Vibrio hollisae. No cross-reactions between V. vulnificus and V. hollisae or V. damsela were observed.
Optimization of in situ hybridization for detection of viral genomes in cultured cells on 96-microwell plates: a cytomegalovirus model
Authors: Mougin C , Bassignot A , Coaquette A , Bourgeois A , Lab M
Published: January 2020

In situ hybridization (ISH) for identification of infectious replicative cytomegalovirus (CMV) in cell culture microplates (96 microwells) infected by clinical specimens was tested by using a biotin-labeled DNA probe and an avidin-alkaline phosphatase conjugate. A total of 395 specimens were examined by using ISH and a monoclonal antibody (MAb) specific for an early antigen of CMV. Of 47 specimens that gave a positive signal for CMV by ISH, 33 were confirmed virus positive by MAb staining. Of 141 blood samples tested, 4.96% were positive by ISH, and 0.7% were positive by the MAb technique. ISH shows 40% more sensitivity than MAb staining. This technique should be widely applicable for the specific identification of viral isolates (e.g., herpesvirus, myxovirus, paramyxovirus, and enterovirus) in cell culture 96-microwell microplates, thereby making it feasible to screen a larger number of samples than is possible with classical methods using conventional culture tubes, shell vials, or 24-well plates.
Adsorption and biotin-streptavidin amplification in serologic tests for diagnosis of Lyme borreliosis
Authors: Magnarelli L A , Anderson J F
Published: January 2020

Serum samples from persons with Lyme borreliosis, periodontitis, or acute necrotizing ulcerative gingivitis were analyzed by an enzyme-linked immunosorbent assay (ELISA) with and without adsorption and amplification procedures. When biotin and streptavidin reagents were used as an amplification procedure in ELISA without the use of commercially prepared sorbent (Treponema phagedenis biotype Reiter), sensitivity increased. Of the 85 serum samples collected from persons with erythema migrans but no detectable antibodies to Borrelia burgdorferi by standard ELISA, 17 (20%) were reactive after amplification. Adsorption of serum samples with a 1:10 dilution of T. phagedenis biotype Reiter sorbent used in conjunction with amplified ELISA also improved the sensitivity of this method. However, cross-reactivity could not be completely eliminated. An adsorbed-amplified ELISA may be helpful in the diagnosis of Lyme borreliosis in the laboratory, particularly during early weeks of infection, when antibodies to B. burgdorferi can be present at a low concentration.
Lyme disease assay which detects killed Borrelia burgdorferi
Authors: Callister S M , Schell R F , Lovrich S D
Published: January 2020

We developed an in vitro assay showing that Borrelia burgdorferi organisms were killed by serum from patients with Lyme disease. Twenty of 20 Lyme disease serum samples caused B. burgdorferi killing in a range of 36 to 99% compared with the mean number of viable spirochetes when sera from 10 healthy individuals were used. The percentage of killing of B. burgdorferi increased with convalescent serum from patients with early Lyme disease. The borreliacidal activity was detectable in some sera diluted 640-fold and was abrogated after treatment with anti-human immunoglobulin G. In contrast, pooled or individual normal human serum did not cause a decrease in the number of viable B. burgdorferi. Borreliacidal activity was also not detected in sera from patients with relapsing fever, rocky mountain spotted fever, syphilis, mononucleosis, rheumatoid factor, or DNA antibodies. Our results show that borreliacidal activity can be used as a specific serodiagnostic test for detecting Lyme disease.
Pneumonia and empyema infection associated with a Bacillus species that resembles B. alvei
Authors: Coudron P E , Payne J M , Markowitz S M
Published: January 2020

An organism resembling Bacillus alvei was isolated from the lung and pleural fluid of an immunocompetent patient. The isolate differed from the type strain of B. alvei in its ability to reduce nitrate and its inability to produce dihydroxyacetone and acetylmethylcarbinol. The isolate was resistant to ciprofloxacin and showed intermediate susceptibility to vancomycin.
Reproducibility of tolerance tests that are useful in the identification of campylobacteria
Authors: On S L , Holmes B
Published: January 2020

Twenty type or other reference strains, each representing a different Campylobacter, Helicobacter, or Arcobacter taxon, were used to assess the reproducibility of 25 phenotypic tests that are used in the identification of such organisms. Twenty-two of the tests depended on growth inhibition, and each of these tolerance tests was performed by using three different basal media. Although the overall reproducibility of the tests with each basal medium exceeded 89%, the proportion of strains that were able to grow in a reproducible manner on the basal media varied from 100% for blood agar and 50% for nutrient agar to 5% for brucella agar. In general, test reproducibility was highest with the basal medium that supported the most luxuriant growth. For the majority of tests, the basal medium which gave the optimum reproducibility could be determined.
Antibody levels and in vitro lymphoproliferative responses to Streptococcus pyogenes erythrogenic toxin A and mitogen of patients with rheumatic fever
Authors: Bahr G M , Yousof A M , Behbehani K , Majeed H A , Sakkalah S , Souan K , Jarrad I , Geoffroy C , Alouf J E
Published: January 2020

We investigated the in vitro lymphoproliferative responses to a streptococcal mitogen and erythrogenic toxin A of children with acute rheumatic fever (ARF) and patients with chronic rheumatic heart disease (CRHD). Antibody levels to the streptococcal products were also analyzed in the sera of those with ARF or chronic rheumatic heart disease as well as in the sera of children with streptococcal pharyngitis or poststreptococcal glomerulonephritis. Our results demonstrated that the individuals had depressed lymphoproliferative responses during the active stage of rheumatic fever. The depressed responses were not found either to be induced by time-sensitive mitogen-specific suppressor cells or to be related to a dose-response phenomenon. On the other hand, antibody levels to the extracellular mitogens were significantly elevated in the sera of children with ARF compared with the levels in the rest of the groups. The hyperresponsiveness noted among children with ARF was found to be at a quantitative level and was not due to recognition of more epitopes, as determined by Western blotting (immunoblotting). The profile of immune responsiveness in children with ARF to the streptococcal extracellular mitogens is discussed in relation to the pathogenesis of disease.
Rapid, low-technology field- and laboratory-applicable enzyme-linked immunosorbent assays for immunodiagnosis of Schistosoma mansoni
Authors: Rossi C L , Tsang V C , Pilcher J B
Published: January 2020

Simple and rapid polystyrene- and nitrocellulose-based enzyme-linked immunosorbent assays were developed for detecting antibodies against adult Schistosoma mansoni microsomal antigens. The polystyrene test uses the Nunc Immuno Stick System. A single dilution of the antibody source being tested, the conjugate, and the substrate (3,3',5,5'-tetramethylbenzidine) are placed in tubes. Dried, antigen-coated polystyrene sticks are then exposed to the reagents by immersion. Once the sticks are sensitized, an entire assay can be completed in 8 min. Positive reactions result in a rich blue color in the substrate tube and can be distinguished with the naked eye. In the nitrocellulose-based test, a nitrocellulose sheet with antigen drawn in a line by pen is cut to produce identical strips. The ligand-binding steps and washings are performed in the troughs of incubation trays. The exposure times required for a single dilution of the antibody source being tested, the conjugate, and the substrate (3,3'-diaminobenzidine) are 5 min, 5 min, and 7.5 min, respectively. Once sensitized strips are available, an entire assay can be run in 50 min. Both techniques can assay serum or whole blood. The characteristics of polystyrene- and nitrocellulose-based techniques allow them to be used successfully in field studies and in minimally equipped laboratories.
Identification of Mycobacterium gordonae from culture by the Gen-Probe Rapid Diagnostic System: evaluation of 218 isolates and potential sources of false-negative results
Authors: Walton D T , Valesco M
Published: January 2020

The Mycobacterium gordonae Rapid Diagnostic System (Gen-Probe, Inc., San Diego, Calif.) was evaluated for sensitivity and specificity as well as for its application in the mycobacteriology laboratory. An 125I-labeled cDNA probe complementary to rRNA was employed. Hybridization of greater than or equal to 10% was considered positive. A total of 218 mycobacterial isolates, including 159 isolates of M. gordonae, were tested. Under optimum conditions, the specificity and sensitivity of the probe were 100 and 98.7%, respectively. A number of discrepancies were observed between the probe and conventional biochemical results in one laboratory. Further studies, designed to resolve these discrepancies, revealed a number of potential technical pitfalls. Hybridization incubation temperatures that varied from the manufacturer's recommended optimum, culture suspensions below the density of a no. 1 McFarland nephelometer standard, and extended storage times of culture suspension all adversely affected the final hybridization values. Additionally, it was determined that in one laboratory incorrect functioning of the sonicator caused false-negative hybridization values. The manufacturer's recommendations should be strictly followed, and the performance of the sonicator should be checked on a scheduled basis. Results show that the probe will allow fast and accurate identification of M. gordonae, thus eliminating time-consuming biochemical testing of this organism.
Diagnostic potential of baculovirus-expressed rubella virus envelope proteins
Authors: Seppänen H , Huhtala M L , Vaheri A , Summers M D , Oker-Blom C
Published: January 2020

The envelope glycoproteins E1 and E2 of rubella virus were abundantly expressed in Spodoptera frugiperda Sf9 insect cells by using a baculovirus expression vector. The recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral E1 and E2 proteins, and a polyprotein precursor (molecular mass, 90 to 95 kDa). The antigen was reactive with human convalescent-phase sera in immunoblot analysis, and the reactivity correlated well (r = 0.861) with that of a whole-virus antigen when tested by EIA by using a total of 106 rubella virus immunoglobulin G-positive and -negative serum specimens. When the sera from patients with recent rubella virus infection were tested with the recombinant glycoproteins by EIA, the correlation was not as close (r = 0.690). However, all of the 26 serum specimens were reactive with the recombinant antigen. The results demonstrate that these bioengineered antigens have a potential for use in routine diagnostic assays of rubella virus immunity and recent infection.
Normally saprobic cryptococci isolated from Cryptococcus neoformans infections
Authors: Krajden S , Summerbell R C , Kane J , Salkin I F , Kemna M E , Rinaldi M G , Fuksa M , Spratt E , Rodriguez C , Rodrigues C
Published: January 2020

We report two cases in which Cryptococcus laurentii was isolated from surgically resected pulmonary lesions but the cryptococcal cells is tissue reacted positively with a specific fluorescent antibody (FA) conjugate for Cryptococcus neoformans. Both patients had no apparent host defense defects. In both cases, multiple cryptococcal isolates were obtained from tissue, and yeastlike cells consistent with C. neoformans were seen in direct histology. The isolates were identified by assimilation patterns and standard procedures including phenoloxidase reactions. Since C. laurentii was consistently isolated by using stringent procedures, it was considered unlikely that the fungus represented surgical or laboratory contamination. Its presence may be the result of dual infection not detected by FA, but other possible explanations exist. The results show the value of the FA test in diagnostic mycology and call into question previous reports of cryptococci other than C. neoformans as agents of infection.
Characterization of murine monoclonal antibodies against serogroup B salmonellae and application as serotyping reagents
Authors: Tsang R S , Chan K H , Lau N W , Choi D K , Law D K , Ng M H
Published: January 2020

Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other O serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.
Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP
Authors: Jackson M P
Published: January 2020

A technique has been developed for the detection of Shiga toxin- and Shiga-like toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli.
Culture adaptation and characterization of group A rotaviruses causing diarrheal illnesses in Bangladesh from 1985 to 1986
Authors: Ward R L , Clemens J D , Sack D A , Knowlton D R , McNeal M M , Huda N , Ahmed F , Rao M , Schiff G M
Published: January 2020

Group A rotaviruses collected between 1985 and 1986 during comprehensive surveillance of treated diarrheal episodes occurring in a rural Bangladesh population were culture adapted and characterized by electropherotype, serotype, and subgroup. Of 454 episodes of rotavirus-associated diarrhea, rotaviruses were culture adapted from 381 (84%), and 335 contained 11 electrophoretically identical segments in unpassaged and cultured preparations. These 335 comprised 69 different electropherotypes with between 1 (32 isolates) and 79 representatives. The persistence of specific rotavirus strains within the study population, as defined by the detection of viruses with particular electropherotypes, was generally limited to a period of only a few months. All 335 isolates were serotyped by neutralization with hyperimmune antisera to prototype rotavirus strains representative of serotypes 1 to 4, i.e., Wa, DS-1, P, and ST-3. It was found that 80, 48, 119, and 88 isolates belonged to serotypes 1 to 4, respectively. The concentrations of hyperimmune antisera required to neutralize these isolates, however, were at least threefold greater than those needed to neutralize the homologous strains. Therefore, the isolates appeared to have altered neutralization epitopes from their prototype strains. Furthermore, the serotype 4 isolates were consistently shown to be much more closely related to the serotype 4B VA70 strain than the serotype 4A ST-3 strain. All but two isolates identified as serotypes 1, 3, or 4 had long electropherotypes and were subgroup II, and all but one serotype 2 isolate were subgroup I and had short electropherotypes. The three disparate strains appeared to be genetic reassortants. Evidence is presented that dual infections required for reassortant formation were not uncommon. Thus, formation of multiple reassortants may have been a cause for the observed rapid shift in viral strains within the study population.