bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle
Authors: Montenegro M A , Bülte M , Trumpf T , Aleksić S , Reuter G , Bulling E , Helmuth R
Published: January 2020

Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1- and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e., O22, O39, O82, O91, O113, O116, O126, and O136. Two isolates from different bulls were identified as serotype O157:H7. Results obtained in this study indicate that cattle may be an important source of verotoxigenic E. coli involved in human disease.
Production of Shiga-like toxin among Escherichia coli strains and other bacteria isolated from diarrhea in São Paulo, Brazil
Authors: Giraldi R , Guth B E , Trabulsi L R
Published: January 2020

An elevated level of Shiga-like toxin I (SLT-I) production was found in 1 of 466 Escherichia coli strains studied. Among the 34 sonic lysates obtained from classical enteropathogenic E. coli, 5 produced SLT-I. The Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Klebsiella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Yersinia, and Vibrio strains also studied were not SLT producers, except for a Shigella dysenteriae type 1 strain. Although SLT-I-producing E. coli strains were isolated from diarrhea, they seem to be an uncommon cause of disease in children less than 1 year old in our community.
Laboratory identification of adenoviruses associated with gastroenteritis in Canada from 1983 to 1986
Authors: Brown M
Published: January 2020

A retrospective analysis of adenovirus serotypes associated with gastroenteritis involved the examination of 143 stool specimens collected between 1983 and 1986 from symptomatic patients whose stools were positive for adenovirus by electron microscopy. The virus isolates obtained from 140 of the specimens were typed according to the SmaI cleavage pattern of the viral DNA and by neutralization with specific antisera. The predominant types were adenovirus type 31 (Ad31) (18%), Ad40 (16.9%), and Ad41 (38%), which together accounted for more than 70% of the isolates. The remaining virus isolates were typed as Ad1, 2, 3, 5, 7, and 12. DNA restriction analysis proved to be better than serum neutralization for identification of the enteric adenovirus serotypes in stool specimens. HindIII cleavage identified four Ad41 variants, none of which had a HindIII restriction pattern identical to that of the prototype strain Tak. Over the time period of the study, the incidence of Ad40 showed an overall decrease accompanied by an increased incidence of Ad41, while the incidence of Ad31 was relatively stable.
A galactosyl(alpha 1-3)mannose epitope on phospholipids of Leishmania mexicana and L. braziliensis is recognized by trypanosomatid-infected human sera
Authors: Avila J L , Rojas M
Published: January 2020

An immunoglobulin M antibody reactive with galactosyl(alpha 1-3)mannose [Gal(alpha 1-3)Man] residues present on phospholipids extracted from Leishmania mexicana and L. braziliensis was found to be present in high titer in the serum of every normal individual studied. Periodate oxidation, acid hydrolysis, or acetylation suppressed immunoreactivity, suggesting that an oligosaccharide chain was responsible for antibody binding. Interaction occurs only with alpha-Gal terminal residues, since treatment of purified glycophospholipids with alpha-galactosidase but not with beta-galactosidase abolished it. Antibody bound to galactosyl(alpha 1-3)galactose-linked synthetic antigens but did not bind to the same residues present in rabbit, rat, and guinea pig erythrocytes or in murine laminin. Antigen-antibody binding was strongly blocked with Gal(alpha 1-3)Man and Gal(beta 1-4)Man. These results plus inhibition studies with several oligosaccharides suggest that they are indeed different from antibodies against the galactosyl(alpha 1-3)galactose residue. Anti-Gal(alpha 1-3)Man antibody values were significantly elevated in 89% of patients with diffuse cutaneous leishmaniasis, 84% of patients with localized cutaneous leishmaniasis, 69% of patients with mucocutaneous leishmaniasis, and 44 and 62% of patients with Trypanosoma cruzi or T. rangeli infection, respectively, but not in patients with 15 other different infectious and inflammatory diseases. Anti-Gal(alpha 1-3)Man antibody readily absorbed to American Leishmania and Trypanosoma culture forms, suggesting a surface membrane localization of reactive epitope. Gal(alpha 1-3)Man-bearing glycophospholipid was easily extracted from American Leishmania promastigotes and T. cruzi trypomastigotes as well as from American Trypanosoma culture forms. The possibility that this antibody arises against parasitic glycophospholipid-linked Gal(alpha 1-3)Man terminal residues is proposed.
Evaluation of Syva enzyme immunoassay for detection of Chlamydia trachomatis in genital specimens
Authors: Gaydos C A , Reichart C A , Long J M , Welsh L E , Neumann T M , Hook E W , Quinn T C
Published: January 2020

Detection of Chlamydia trachomatis infection was evaluated by culture and a new Syva enzyme immunoassay (EIA) in 1,012 patients at two Baltimore, Md., sexually transmitted disease clinics. The overall chlamydia prevalence determined by culture was 12%. For 506 fresh cervical and urethral specimens, the sensitivity of Syva EIA was 90% and its specificity was 94% compared with culture. Discordant Syva EIA results were further evaluated by staining the sediment in centrifuged culture transport media and Syva EIA transport tubes with a fluorescent monoclonal antibody to C. trachomatis to detect elementary bodies. Reanalysis of the data after use of this technique to resolve discordant results increased sensitivity and specificity to 92 and 96%, respectively. A subsample of 307 fresh cervical specimens was also tested in a three-way comparison using Abbott Chlamydiazyme, Syva EIA, and culture. In this sample, compared with culture, the sensitivity and specificity of Syva EIA were 87 and 95%, respectively, and for Chlamydiazyme they were 77 and 98%, respectively. Syva EIA is a 4-h, easy-to-perform enzyme-linked immunosorbent assay which has a high sensitivity with fresh genital specimens and offers an excellent alternative to culture.
Longitudinal survey in an endemic region of plasma soluble interleukin-2 receptor and antibody levels in Plasmodium falciparum malaria
Authors: Chumpitazi B F , Peyron F , Simon J , Boudin C , Sheick-Zakiuddin I , Picot S , Ambroise-Thomas P
Published: January 2020

A survey involving 81 individuals living in Dafinso and Vallée du Kou no. 4 (near Bobo-Dioulasso), Burkina Faso, was performed in June 1987, August to September 1987, and January 1988, respectively, at the beginning of, during, and after the transmission season of malaria. The clinical longitudinal study during the transmission period allowed us to define three different groups in terms of both age and occurrence of malaria attack (5,000 Plasmodium falciparum per mm3 of blood and axillary fever of greater than 37.7 degrees C) as follows: group 1, persons less than or equal to 15 years old who had at least one malaria attack during the transmission period; group 2, individuals less than or equal to 15 years old who did not have any malaria attacks; and group 3, individuals considered to be protected (adults greater than 15 years old with no malaria attacks). Soluble interleukin-2 receptor (sIL-2R) levels were found to be significantly increased (P less than 0.001) in the first two groups (1,047 +/- 481 U/ml [mean +/- standard deviation]) as compared with the adult group (605 +/- 307 U/ml). Considering all the groups, no significant difference was observed between observation periods. Levels of sIL-2R were inversely correlated (r = -0.39, n = 237, P less than 0.01) with age (range, 4 to 67 years). Negative correlations were also noticed between the levels of sIL-2R and those of antibodies to somatic antigen of P. falciparum (immunoglobulin G [IgG] class [r = -0.33, n = 237, P less than 0.01] and IgM class [r = -0.20, n = 237, P less than 0.05]). IgG antibody levels to somatic antigen were correlated with age, but IgM antibody levels to somatic antigen were not. The possible role played by sIL-2R in effector mechanisms against malaria is discussed.
Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers
Authors: Albert J , Fenyö E M
Published: January 2020

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.
Trichosporon beigelii, an emerging pathogen resistant to amphotericin B
Authors: Walsh T J , Melcher G P , Rinaldi M G , Lecciones J , McGough D A , Kelly P , Lee J , Callender D , Rubin M , Pizzo P A
Published: January 2020

Trichosporon beigelii caused fatal disseminated infections that were resistant to amphotericin B in two granulocytopenic patients. In vitro susceptibility studies demonstrated that both index strains of T. beigelii were inhibited but not killed by amphotericin B at achievable concentrations in serum. The minimum lethal concentration for both isolates was greater than or equal to 18 micrograms/ml. Five of seven other isolates were found to have a similar pattern of amphotericin B resistance. The fact that the minimum lethal concentration of T. beigelii was many times greater than its MIC was consistent with a resistance pattern of tolerance. We concluded that T. beigelii may be resistant in vitro to amphotericin B and that this in vitro resistance was correlated with refractory, disseminated trichosporonosis in granulocytopenic patients. T. beigelii should be included in the expanding list of amphotericin B-resistant fungi.
Rabbit model to evaluate enterovirulence of Bacteroides fragilis
Authors: Myers L L , Shoop D S , Collins J E
Published: January 2020

Enterotoxigenic Bacteroides fragilis elicited fluid accumulation in the intestinal tract and exfoliation of epithelial cells in the proximal colon of the 2-week-old rabbit. The rabbit model was employed to screen isolates of B. fragilis for enterovirulence and to study the pathogenesis of disease caused by enterotoxigenic B. fragilis.
Buffered charcoal-yeast extract medium for the isolation of brucellae
Authors: Raad I , Rand K , Gaskins D
Published: January 2020

A patient with chronic osteomyelitis caused by Brucella abortus had negative agglutination titers. Because of a superimposed staphylococcal infection that resulted in the overgrowth of this organism on nonselective media, brucellae were isolated only on a selective buffered charcoal-yeast extract (BCYE) agar. Sixteen strains of various Brucella species were inoculated on BCYE agar; selective BCYE agar with polymyxin, anisomycin, and cefamandole; and brucella blood agar. The growth and recovery rates on the three media tested were comparable for 14 strains. BCYE agar with polymyxin, anisomycin, and cefamandole may be useful as a selective medium for the isolation of brucellae.
Evaluation of the Captia enzyme immunoassays for detection of immunoglobulins G and M to Treponema pallidum in syphilis
Authors: Lefevre J C , Bertrand M A , Bauriaud R
Published: January 2020

Two new enzyme-linked immunosorbent assays (ELISA), one for the measurement of immunoglobulin G (IgG) (Captia Syphilis-G) and one for the measurement of IgM (Captia Syphilis-M), were evaluated for detecting antibodies to Treponema pallidum. Serum samples from 169 patients, 96 with various stages of untreated syphilis, 63 with treated syphilis, and 10 who were noninfected, were investigated. All sera were also examined by traditional treponemal and cardiolipin tests and by the fluorescent treponemal antibody absorption (FTA-ABS) test for 19S(IgM). The overall sensitivity of Captia Syphilis-G was 98.3%. The IgG ELISA was very sensitive (100%) in all stages of untreated syphilis, except in primary syphilis (82%). In all diagnostic groups of syphilis, the reactivity of Captia Syphilis-M was similar to that of the 19S(IgM) FTA-ABS test, except in reinfections, in which the IgM capture ELISA was less sensitive. False-positive IgM capture ELISA results were not found in the 10 neonates born to mothers adequately treated for syphilis. However, of six serum samples containing rheumatoid factor, two were reactive in the Captia Syphilis-M test but not in the 19S(IgM) FTA-ABS test. This indicated that the specificity of the IgM capture ELISA was not absolute. All serum samples from treated patients were reactive in the IgG ELISA, but only 15 samples were reactive in the IgM capture ELISA, which appeared to be as effective as the 19S(IgM) FTA-ABS test in monitoring the effect of treatment. Simultaneous measurement of IgG and IgM antibodies for T. pallidum by the Captia immunoassays appears to be an efficient and simple method for confirming the diagnosis of syphilis as well as for indicating whether active disease is present.
Comparison of two enzyme immunoassays to culture for the diagnosis of chlamydial conjunctivitis and respiratory infections in infants
Authors: Hammerschlag M R , Roblin P M , Gelling M , Worku M
Published: January 2020

Data are limited for the performance of enzyme immunoassays for the detection of Chlamydia trachomatis in conjunctival and nasopharyngeal specimens from infants. The only available data are for one assay, Chlamydiazyme (Abbott Diagnostics). The purpose of this study was to compare a new enzyme immunoassay, Pathfinder (Kallestad Diagnostics), with Chlamydiazyme and culture for the diagnosis of chlamydial conjunctivitis and pneumonia in infants. Pathfinder differs from Chlamydiazyme in that it uses a monoclonal antibody directed against the chlamydial lipopolysaccharide in addition to a polyclonal antichlamydial antibody. Triplicate conjunctival and nasopharyngeal specimens were obtained from 97 infants with conjunctivitis, and additional nasopharyngeal specimens were obtained from 14 infants with suspected chlamydial pneumonia (total, 111 nasopharyngeal specimens). Twenty-nine (30%) of the conjunctival specimens from infants with conjunctivitis and four (28.6%) of the nasopharyngeal specimens from the infants with pneumonia were positive for C. trachomatis by cell culture. The sensitivities, specificities, and positive and negative predictive values for Pathfinder for conjunctival specimens were 96.6, 98.5, 96.6, and 98.5%, respectively. The results for Chlamydiazyme were 96.6, 100, 100, and 98.6%, respectively. For nasopharyngeal specimens, the results for Pathfinder were 77.8, 94.6, 73.7, and 95.7%, respectively. The results for Chlamydiazyme were 66.7, 95.7, 75, and 93%, respectively. Pathfinder and Chlamydiazyme appeared to perform equivalently for the detection of C. trachomatis in both eye and nasopharyngeal specimens from infants with chlamydial conjunctivitis and pneumonia.
Interlaboratory and intralaboratory comparisons of indirect immunofluorescence assays for serodiagnosis of Lyme disease
Authors: Lane R S , Lennette E T , Madigan J E
Published: January 2020

A conventional indirect immunofluorescence assay (IFA) and an anticomplement indirect immunofluorescence assay (ACIF) for detecting serum antibodies to Borrelia burgdorferi in humans were evaluated during a prevalence survey in northern California. Sera obtained from 119 current or former residents of an area in which Lyme disease is endemic were split and tested by the IFA in two laboratories and the ACIF in a third. The seropositivity rate ranged from 15 to 20% with 88 to 93% agreement among laboratories. Interlaboratory agreement was statistically highly significant in each of the three pairwise comparisons and was positively associated with clinical manifestations of Lyme disease. Intralaboratory agreement ranged from 93 to 96% in two laboratories and was also statistically highly significant. Immunoblotting confirmed 100 of 101 of the nondiscrepant immunofluorescence test results and likewise was positively correlated with the degree of interlaboratory agreement. The ACIF was found to be a highly specific test (100% specificity) with a much lower cutoff titer (1:8) than the conventional IFA (determined to be 1:128 or 1:256 in two laboratories) for detecting antibodies to B. burgdorferi. It also appeared to be more sensitive (80 versus 68%) than the IFA as determined by comparative immunoblotting, though the absolute sensitivity of the ACIF for serodiagnosis of early Lyme disease has yet to be determined. Significant serologic cross-reactivity was demonstrated between B. burgdorferi, Borrelia coriaceae, and Borrelia hermsii by the IFA, which may confound spirochetal serosurveys in California where all three spirochetes are known to coexist.
Evidence for a group-specific enteroviral antigen(s) recognized by human T cells
Authors: Beck M A , Tracy S M
Published: January 2020

Human peripheral blood mononuclear cells from 15 normal, healthy adult volunteers proliferated in vitro against a panel of enteroviral antigens, including coxsackievirus B3, coxsackievirus B2, coxsackievirus B6, coxsackievirus A16, and poliovirus 1. No proliferation against the cardiovirus encephalomyocarditis virus occurred. Lymphocytes obtained from cord blood drawn from seven neonates were uniformly nonresponsive to enteroviral antigens. Although serum neutralization antibody titers indicated different exposure histories of the volunteers, only one had a titer against coxsackievirus B6, a rare isolate in the United States. The peripheral blood mononuclear cells from each volunteer responded in vitro to each enterovirus tested even though not all individuals had serum neutralizing antibody against each virus. The predominant cell type responding in vitro was the CD4+ T cell. Denaturation of viral antigen by Formalin did not prevent the recognition of the common group antigen by the T cells, indicating that noninfectious virus can also serve as antigen. These data demonstrate that human T cells recognize a common enterovirus group antigen(s).
Presumptive identification of Clostridium difficile by detection of p-cresol in prepared peptone yeast glucose broth supplemented with p-hydroxyphenylacetic acid
Authors: Sivsammye G , Sims H V
Published: January 2020

Prereduced, anaerobically sterilized peptone yeast glucose broth was supplemented with p-hydroxyphenylacetic acid and used for the presumptive identification of Clostridium difficile. Two hundred eighty-two organisms were grown in this medium for 18 h and tested for p-cresol production by gas-liquid chromatography. All 49 stock and reference strains of C. difficile and 19 organisms confirmed as C. difficile produced p-cresol. p-Cresol was not produced by 53 negative control or 161 test organisms. The system was convenient and effective.
Specificity of DNA probes for the detection of toxigenic Pasteurella multocida subsp. multocida strains
Authors: Kamps A M , Buys W E , Kamp E M , Smits M A
Published: January 2020

Five DNA probes directed against different regions of the gene that encodes the dermonecrotic toxin of Pasteurella multocida subsp. multocida were examined for their ability to identify toxigenic P. multocida subsp. multocida strains. The specificities of the probes were studied with 96 strains of P. multocida subsp. multocida and 22 strains of 11 other bacterial species. Results of colony hybridization assays using these probes indicated that two of the five probes have potential diagnostic value.
Detection and identification of Mycobacterium tuberculosis by DNA amplification
Authors: Pao C C , Yen T S , You J B , Maa J S , Fiss E H , Chang C H
Published: January 2020

The polymerase chain reaction (PCR) was used to identify mycobacterial DNA sequences in uncultured clinical specimens. Two oligonucleotide primers derived from the sequence of a gene that codes for the 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA from all 11 species of mycobacteria tested. Amplified DNAs of nontuberculosis mycobacteria were found to be approximately 20 to 40 bases shorter than those from M. tuberculosis and Mycobacterium bovis BCG. DNA equivalent to that present in as few as 40 M. tuberculosis cells either alone or in the presence of DNA equivalent to that in 10(6) human cells could be detected. Results from analysis of cultured bacteria and clinical specimens showed PCR was sensitive and specific both in detecting mycobacteria and in differentiating M. tuberculosis and BCG from other species of mycobacteria. The PCR method with the primers reported here may become a useful tool in the early and rapid detection of mycobacterial infections in uncultured clinical specimens.
Immunodiffusion test for serodiagnosing subcutaneous zygomycosis
Authors: Kaufman L , Mendoza L , Standard P G
Published: January 2020

Culture filtrate antigens of Basidiobolus ranarum and Conidiobolus coronatus were analyzed by immunodiffusion (ID) with homologous rabbit antisera. B. ranarum and C. coronatus were each found to have five specific antigens. Results of tests with heterologous antisera indicated that all of the species shared at least one antigen. ID tests incorporating the specific precipitin bands as references were developed for detection of basidiobolomycosis and conidiobolomycosis. These tests were performed with sera from humans and horses with proven basidiobolomycosis and conidiobolomycosis as well as with control sera from humans and animals with and without heterologous mycotic and oomycotic infections. Only sera from cases of basidiobolomycosis and conidiobolomycosis produced lines of identity with the reference precipitates of B. ranarum and C. coronatus, respectively. The ID tests were found to be completely sensitive and specific for determining the etiology of zygomycosis caused by these two species. In addition they appeared useful for monitoring resolution of the infections.
Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli
Authors: Lopez-Vidal Y , Svennerholm A M
Published: January 2020

Monoclonal antibodies (MAbs) against the different coli surface antigens CS1, CS2, and CS3 of colonization factor antigen II (CFA/II) of enterotoxigenic Escherichia coli (ETEC) were generated by fusing F/O myeloma cells with spleen cells from BALB/c mice immunized with different preparations of purified CFA/II. Five hybrids that produced antibodies specific for CS1, CS2, or CS3 in high titer were cloned and propagated. All the anti-CS MAbs were of the immunoglobulin G1 isotype, and all gave single precipitation lines in immunodiffusion tests when reacting with CFA/II-positive E. coli extracts containing the corresponding CS factor. The binding of all the MAbs to solid-phase-bound CFA/II could be completely inhibited by purified CFA/II containing the corresponding CS factor. However, whereas one MAb against CS3 was inhibited by all of 18 different CFA/II-positive strains tested, another anti-CS3 MAb was inhibited by bacteria expressing CS1 and CS3 (CS1 + CS3 strains) or CS3 alone but not by CS2 + CS3 strains, suggesting antigenic differences in CS3 when expressed by different strains. Use of the anti-CS MAbs in slide agglutination, immunodiffusion, or a CFA inhibition enzyme-linked immunosorbent assay revealed differences in the relative distribution of the various CS factors of CFA/II in clinical ETEC isolates from different geographic areas. By using the anti-CS MAbs in an enzyme-linked immunosorbent assay-nitrocellulose replica method, CFA/II-positive colonies could be detected in stool cultures from infected animals without prior isolation of the ETEC organisms.
Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection
Authors: Yamamoto M , Black J B , Stewart J A , Lopez C , Pellett P E
Published: January 2020

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.
Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa
Authors: Kelly N M , MacDonald M H , Martin N , Nicas T , Hancock R E
Published: January 2020

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.
High-performance liquid chromatography patterns of mycolic acids as criteria for identification of Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium smegmatis
Authors: Butler W R , Kilburn J O
Published: January 2020

Rapidly growing mycobacteria of clinical significance were identified by mycolic acids detected with high-performance liquid chromatography. Mycolic acids from whole cells were extracted, derivatized, and detected by a modified high-performance liquid chromatography procedure in less than 3 h. Use of an internal standard allowed differentiation of Mycobacterium chelonae and Mycobacterium fortuitum by comparison of relative retention times. Peak height ratios were used for subidentification of M. chelonae strains; however, M. fortuitum and Mycobacterium smegmatis could not be separated by this system.
Evaluation of two commercial procedures for rapid identification of Neisseria gonorrhoeae using a reference panel of antigenically diverse gonococci
Authors: Boehm D M , Bernhardt M , Kurzynski T A , Pennell D R , Schell R F
Published: January 2020

Two commercial tests for the rapid identification of Neisseria gonorrhoeae were evaluated. Two hundred seventy-nine organisms were tested, including 202 strains of N. gonorrhoeae. The Syva MicroTrak test results were less subjective but required a fluorescence microscope. The Phadebact Monoclonal GC OMNI Test required modification of the manufacturer's interpretive instructions in order to avoid cross-reactions, but it was a practical test. Specificities of both tests were 100%. Sensitivities of the Phadebact Monoclonal GC OMNI and Syva MicroTrak tests were 100% and approximately 100%, respectively.
Comparison of alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test for identification of Shigella strains
Authors: Panda C S , Riley L W , Kumari S N , Khanna K K , Prakash K
Published: January 2020

We compared an alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test to determine the sensitivity and specificity of the probe in detecting virulent Shigella strains. The probe hybridized with all 52 Sereny-test-positive strains (sensitivity, 100%) and 4 of 21 Sereny-test-negative strains (specificity, 81%). The probe did not hybridize with any of the Sereny-test-negative S. dysenteriae type 1 strains. This nonradioactive, synthetic probe provides a simple, rapid way to test a large number of strains simultaneously in a field setting, which will contribute to an improved understanding of the epidemiologic patterns of shigellosis in developing countries.
Interlaboratory comparison of titers of antibody to Borrelia burgdorferi and evaluation of a commercial assay using canine sera
Authors: Greene R T , Hirsch D A , Rottman P L , Gerig T M
Published: January 2020

Sixty canine serum samples were sent to 10 different diagnostic laboratories for anti-Borrelia burgdorferi antibody analysis. All laboratories knew of the study prior to receiving the samples. Agreement among all laboratories for all interpretations was 91% (546 of 600 samples). There was complete agreement among all the laboratories for only 32 (53%) of the samples. Most of the disagreements were due to differences reported by either one (15 samples) or two (7 samples) laboratories per sample. When discrepancies in interpretations existed, the interpretation reported by the majority of the laboratories was considered the standard for comparison. One laboratory had no discrepant interpretations from this standard, while the laboratory with the most discrepancies had 16. The median number of discrepancies per laboratory was five. By using pairwise comparisons between each laboratory and the majority standard, eight of the laboratories showed strong agreement and the remaining two showed fair to good agreement. The type of test used (enzyme-linked immunosorbent assay versus indirect immunofluorescence assay) did not appear to influence the number of discrepant interpretations reported. Sera considered to be positive by the majority of the laboratories usually reacted to more than five antigens in immunoblots, with at least three or more of those being intense reactions. For positive samples, reactivity was consistently present in the 60-, 41-, 31-, and 22- or 24-kDa regions. Samples considered negative usually reacted to fewer than three bands, with reactivity usually being faint. A commercially available, rapid dot blot assay showed strong agreement with the majority standard.
Sensitive detection of Treponema pallidum by using the polymerase chain reaction
Authors: Burstain J M , Grimprel E , Lukehart S A , Norgard M V , Radolf J D
Published: January 2020

We have developed a sensitive assay for Treponema pallidum subsp. pallidum (T. pallidum), the agent of veneral syphilis, based upon the polymerase chain reaction (PCR). A 658-bp portion of the gene encoding the 47-kDa membrane immunogen was amplified, and the PCR products were probed by DNA-DNA hybridization with a 496-bp fragment internal to the amplitifed DNA. The assay detected approximately 0.01 pg of purified T. pallidum DNA, and positive results were obtained routinely from suspensions of treponemes calculated to contain 10 or more organism and from some suspensions calculated to contain a single organism. Specific PCR products were obtained for the closely related agent of yaws, Treponema pallidum subsp. pertenue, but not with human DNA or DNAs from other spirochetes (including Borrelia burgdoferi), skin microorganisms, sexually transmitted disease pathogens, and central nervous system pathogens. T. pallidum DNA was detected in serum, cerebrospinal fluids, and amniotic fluids from syphilis patients but not in in nonsyphilitic controls. T. pallidum DNA was also amplified from paraffin-embedded tissue. The diagnosis of syphillis by using PCR may become a significant addition to the diagnostic armamentarium and a valuable technique for the investigation of syphilis pathogenesis.
Evaluation of a simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli which is based on the O antigen
Authors: Mills S D , Congi R V , Hennessy J N , Penner J L
Published: January 2020

A simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens was developed and tested for its applicability as a routine typing method. The assay involves the sensitization of erythrocytes with an antigenic extract and performance of a slide agglutination assay with specific antisera. In order to simplify the typing system to a greater extent, the standard typing antisera were pooled into nine groups for C. jejuni and four groups for C. coli. The five antiserum samples allocated to each pool were selected so that pairs or groups of cross-reacting antisera were included in the same pool. When this system was tested with the serotype reference strains, it was found that, in most cases, a strain reacted in only one pool. The specific serotype of that strain could then be further defined by typing in each of the antisera belonging to that pool. To evaluate the specificity of the simplified method, 246 clinical isolates of C. jejuni and 57 clinical isolates of C. coli were typed at the same time by the standard passive hemagglutination assay and by the rapid slide agglutination system. Although both schemes effectively differentiated isolates and results from both schemes were generally very similar, differences were noted for a few isolates. On the basis of these findings, the simplified procedure may be recommended as an alternative means for serotyping these species for epidemiological purposes.
Identification of Mycobacterium kansasii by DNA hybridization
Authors: Huang Z H , Ross B C , Dwyer B
Published: January 2020

A DNA probe specific for Mycobacterium kansasii was obtained from a plasmid clone library of EcoRI-digested genomic DNA. The probe specifically identified culture-confirmed isolates of M. kansasii and isolates in cultures of environmental water samples. In an attempt to distinguish between isolates of M. kansasii, we used two methods to demonstrate restriction fragment length polymorphisms in the genomic DNA. Both of these methods failed to detect any differences between the isolates. These isolates included the type strain TMC 1201, environmental isolates, and clinical isolates from Australia and the Solomon Islands. This result suggests that the genome of M. kansasii is highly conserved and that genetic divergence within this species in insignificant.
Typing of human polyomavirus JC virus on the basis of restriction fragment length polymorphisms
Authors: Yogo Y , Iida T , Taguchi F , Kitamura T , Aso Y
Published: January 2020

JC virus DNA clones from the urine of nonimmunosuppressed Japanese individuals regularly contain an archetypal regulatory sequence which may have generated various regulatory sequences of JC virus isolates from patients with progressive multifocal leukoencephalopathy (PML). In this study, we established 15 new clones from the urine of Dutch, German, and Taiwanese healthy volunteers and patients. Most of these clones contained regulatory sequences essentially identical to the archetypal regulatory sequence. These clones, along with two representative urine-derived clones in Japan and five clones from the brains of PML patients (four established in the United States and one established in Japan), were analyzed with a number of restriction enzymes. We found nine restriction fragment length polymorphisms by which all clones were classified into either of the two types, A and B. Type A contained only clones from the West, while type B contained some from the West and all from eastern Asia. Each type contained both urine-derived and PML-derived clones. Furthermore, there was a close relationship between some urine-derived clones and some PML-derived clones in restriction site mapping analysis. These findings support the adaptation hypothesis which has been postulated to explain the genesis of PML-type JC viruses.
Advantages of a human immunodeficiency virus type 1 (HIV-1) persistently infected HeLa T4+ cell line for HIV-1 indirect immunofluorescence serology
Authors: Forghani B , Hurst J W , Chan C S
Published: January 2020

A HeLa T4+ cell line persistently infected with human immunodeficiency virus type 1 (HIV-1) was used in an indirect immunofluorescent antibody assay (IFA) system to explore its potential suitability as an alternative source of viral antigen for confirmatory IFA in HIV serology. In a study of 121 serum samples chosen because they were reactive on repeat examination by enzyme immunoassay but nonspecific by IFA by using HIV-1-infected H9 cells (H9 IFA) or gave discrepant results by enzyme immunoassay and H9 IFA, the specificity and sensitivity of the HeLa T4+ IFA were comparable to those of Western blot (immunoblot), and identification of the true positive samples among these discrepant or nonspecific samples by HeLa T4+ IFA was approximately twice that by H9 IFA. The primary advantages of using the HeLa cell line rather than lymphoid cell lines in IFA are that cells can be grown as a monolayer and that the individual cells are much larger. The cell membrane, cytoplasm, and nucleus are easily discernible; this allows specific and nonspecific staining to be distinguished. At least eight different nonspecific nuclear and cytoplasmic staining patterns were identified in this study by using T4+ cells.
Sensitive analysis of genetic heterogeneity of adenovirus types 3 and 7 in the Soviet Union
Authors: Golovina G I , Zolotaryov F N , Yurlova T I
Published: January 2020

An analysis of adenovirus strains isolated in the Soviet Union from 1976 to 1988 revealed four genome types of adenovirus type 3 (Ad3), i.e., Ad3a4, Ad3a9, Ad3a10, and Ad3a11, and four genome types of adenovirus type 7 (Ad7), i.e., Ad7p, Ad7a, Ad7a(1-5), and Ad7f1, identified with the DNA restriction enzymes BamHI, BglII, and HindIII. Three of them, Ad3a10, Ad3a11, and Ad7f1, are newly discovered. The genetic heterogeneity of adenoviruses was examined with restriction endonuclease Cfr13I with a 4-base recognition cleavage site. Eighteen different restriction patterns were identified among 21 selected Ad3 strains after cleavage of DNA with Cfr13I. Eight different subtypes were identified among 20 Ad7 strains by the same technique. For estimation of the relationships among these genome subtypes, pairwise analyses of comigrating DNA restriction fragments from isolates of Ad3 and Ad7 were done after digestion with Cfr13I or with restriction endonucleases recognizing DNA sequences of 6 bp. Surprisingly, the results were very discrepant.
Novel plasmid combinations in Haemophilus ducreyi isolates from Thailand
Authors: Sarafian S K , Johnson S R , Thomas M L , Knapp J S
Published: January 2020

Thirty isolates of Haemophilus ducreyi collected in Thailand in 1984 were characterized by plasmid content. Three novel plasmids with estimated molecular masses of 1.8, 2.6, and 2.8 MDa were observed in 29 isolates, in addition to the 3.2-, 5.7-, and 7.0-MDa beta-lactamase and 4.4-MDa sulfonamide resistance plasmids. At least three of the seven plasmids were observed in each of the 29 isolates. The number and diversity of plasmids observed in these isolates of H. ducreyi distinguish them from strains previously described.
Diagnosis of invasive candidiasis by detection of mannan antigen by using the avidin-biotin enzyme immunoassay
Authors: Nakamura A , Ishikawa N , Suzuki H
Published: January 2020

The diagnosis of invasive candidiasis was attempted by detection of circulating mannan antigen by using an avidin-biotin-amplified enzyme-linked immunosorbent assay (AB-ELISA), and this method was compared with the conventional culture method. Mannan antigen was detected by AB-ELISA in the sera of 16 (84.2%) of the 19 patients with invasive candidiasis. On the other hand, for 34 immunocompromised candidiasis-free patients, including 8 with aspergillosis or cryptococcosis, mannan antigen was positive during only 1 of the 67 febrile episodes and in the serum of none of the 50 outpatients without infections. The results were also negative for all patients with deep-seated mycoses other than candidiasis. However, the mannan level was low (less than 2.0 ng/ml) in the serum of 63.2% of the patients with invasive candidiasis. The positivity rate of blood cultures was 31.6%, and that of blood cultures and/or cultures of samples from sterile sites combined was 47.4%. The advantages of the diagnosis based on antigen detection by AB-ELISA are considered to be a higher sensitivity and elimination of nonspecific reactions by the introduction of the avidin-biotin system and pretreatment of sera by heating. In addition, it is considered essential for high sensitivity that transient mannan antigenemia be determined frequently so that it is not overlooked. In light of its sensitivity and specificity, this method is considered to be clinically useful in the diagnosis of invasive candidiasis.
Variability in growth of Neisseria polysaccharea on colistin-containing selective media for Neisseria spp
Authors: Anand C M , Ashton F , Shaw H , Gordon R
Published: January 2020

In a prospective survey of 773 healthy schoolchildren in southern Alberta, Canada, Neisseria polysaccharea was isolated from the pharynxes of only 4 (0.5%) subjects, whereas Neisseria lactamica and Neisseria meningitidis were isolated from 110 (14%) and 15 (2%) children, respectively. These strains of N. polysaccharea, together with three other sporadic isolates from Alberta, Canada, were compared with the type strain from France and strains from Spain and Germany. All strains were phenotypically identical, except that the Canadian and German strains, for which the colistin MICs were 1 mg/liter, failed to grow on Thayer-Martin medium (TMM), whereas the type strain and the Spanish strains, for which the colistin MICs were greater than 7.5 mg/liter, were not inhibited. Multilocus enzyme electrophoresis indicated that six distinct electrophoretic types were present among the seven Canadian strains. Our results show that growth on gonococcal selective media which contain colistin is a variable feature of this taxon.
Analysis of the role of flagella in the heat-labile Lior serotyping scheme of thermophilic Campylobacters by mutant allele exchange
Authors: Alm R A , Guerry P , Power M E , Lior H , Trust T J
Published: January 2020

Flagellin mutations originally constructed in Campylobacter coli VC167 (serotype LIO8) by a gene replacement mutagenesis technique (P. Guerry, S. M. Logan, S. Thornton, and T. J. Trust, J. Bacteriol. 172:1853-1860, 1990) were moved from the original host into Campylobacter strains of a number of other Lior serogroups by a natural transformation procedure. This is the first report of the use of this transformation method to transfer a mutated locus among Campylobacter strains. Flagellin mutants were constructed in a number of heat-labile LIO serotypes and were serotyped and analyzed by immunoelectron microscopy with LIO typing antisera. In six cases, isogenic nonflagellated mutants were able to be serotyped in the same serogroup as their parent, and immunogold electron microscopy confirmed that antibodies in the typing antisera bound to components on the surface of both parent and mutant cells. However, in only one case, a strain belonging to serogroup LIO4, was a nonflagellated mutant untypeable, and immunogold electron microscopy showed that antibodies bound to the flagella filament of the parent but not to the cell surface. Furthermore, after introduction and expression as a flagellar filament of a LIO8 flagellin gene in this mutant, the strain could not be serotyped. These results indicate that a nonflagellar antigen is often the serodeterminant in the heat-labile Lior serotyping scheme.
Identification of mycobacteria by high-performance liquid chromatography
Authors: Butler W R , Jost K C , Kilburn J O
Published: January 2020

Mycolic acids extracted from saponified mycobacterial cells were examined as p-bromophenacyl esters by high-performance liquid chromatography (HPLC). Standard HPLC patterns were developed for species of Mycobacterium by examination of strains from culture collections and other well-characterized isolates. Relative retention times of peaks and peak height comparisons were used to develop a differentiation scheme that was 98% accurate for the species examined. A rapid, cost-effective HPLC method which offers an alternative approach to the identification of mycobacteria is described.
Immunoblot studies to analyze antibody to the Rickettsia typhi group antigen in sera from patients with acute febrile cerebrovasculitis
Authors: Hechemy K E , Fox J A , Gröschel D H , Hayden F G , Wenzel R P
Published: January 2020

In 1986, an unusual syndrome of acute febrile cerebrovasculitis in the Piedmont Region of Virginia was reported. All patients had encephalopathy and prior exposure to both a sylvan environment and flea-infested animals. The initial serological studies suggested a rickettsial origin, corroborating clinical, epidemiological, and histopathological findings. Sera from four of five patients were subsequently studied by immunoblotting. Unabsorbed and absorbed sera were tested with electrophoresed and electroblotted Rickettsia typhi, Legionella bozemanii, and Proteus vulgaris OX19 antigens. The unabsorbed sera reacted with all three antigens. The P. vulgaris- and L. bozemanii-absorbed sera reacted with R. typhi only and without significantly less intensity. In contrast, the reactivity of R. typhi-absorbed sera was significantly lower with all three antigens. These results indicate that these patients had specific antibodies to a typhus group antigen. Although our findings suggest that a rickettsia of the typhus group may have caused this syndrome, no definitive diagnosis could be achieved because a rickettsial organism was not isolated.