bluuebunny/crossref_metadata_2025 huggingface dataset:
Nationwide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand
Authors: Sunthadvanich R , Chiewsilp D , Seriwatana J , Sakazaki R , Echeverria P
Published: January 2020

Escherichia coli strains isolated from children with diarrhea were collected from 16 hospitals in different districts in Thailand during 1985 and 1986 and submitted to the National Reference Laboratory. Isolates were identified by serogrouping or as enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) adhesin factor (EAF) E. coli, or Shiga-like-toxin (SLT)-producing E. coli by DNA hybridization. EPEC strains of known serogroups were isolated from 10%, ETEC strains were isolated from 6%, EAF E. coli strains were isolated from 4%, EIEC strains were isolated from less than 1%, and SLT-producing E. coli strains were isolated from none of 393 children with diarrhea. Among 278 children whose ages were recorded, the highest rate of isolation of EAF E. coli was 11% (9 of 85) from children less than 6 months old. ETEC was isolated from 5% (4 of 85) of children less than 6 months old, from 10% (12 of 118) of children 6 to 23 months old, and from 1% (1 of 75) of children greater than 23 months old. EPEC strains of known serogroups were isolated from 18% (15 of 85) of children less than 6 months old, from 11% (13 of 118) of children 6 to 23 months old, and from 9% (7 of 75) of children greater than 23 months old. E. coli strains that hybridized with the EIEC probe were isolated from three children who were 20, 36, and 48 months old. Examining E. coli for hybridization with DNA probes for virulence determinants is a practical way of conducting nationwide surveillance of diarrhea-causing E. coli. Since only 33% (13 of 39) of EPEC serogroups hybridized with the EAF probe and none hybridized with the SLT probes, identification of EPEC by serogroups analysis, followed by serotyping, should continue to be used in the identification of EPEC.
Recovery of vancomycin-resistant gram-positive cocci from children
Authors: Green M , Wadowsky R M , Barbadora K
Published: January 2020

A cross-sectional survey of vancomycin-resistant gram-positive cocci (VRGPC) in the feces of children was initiated after several bacteremic infections with these organisms occurred at our hospital. A selective medium consisting of colistin-nalidixic acid agar, 5% sheep blood, vancomycin (5 mg/liter), and amphotericin B (8 mg/liter) was developed to isolate VRGPC. A single stool specimen submitted to the clinical microbiology laboratory from each of 48 patients was inoculated onto the medium. Plates were incubated at 35 degrees C with 5% carbon dioxide and examined at 24, 48, and 72 h. Susceptibilities were determined by broth microdilution. A total of 14 isolates from 11 of 48 (22%) children were recovered. The density of growth ranged from a single colony to 2+. The VRGPC were identified as Leuconostoc lactis (n = 2), Lactobacillus confusus (n = 4), Enterococcus species (n = 5), and Lactococcus lactis (n = 3). One strain of Lactobacillus confusus was recovered from both the stool and the blood of one of these patients. The MICs of vancomycin were 4 micrograms/ml for one of the isolates, 8 micrograms/ml for four of the isolates, and more than 16 micrograms/ml for the remaining eight isolates. All isolates were susceptible to both penicillin and ampicillin. Only 1 of the 11 children had received prior treatment with vancomycin. We conclude that low concentrations of VRGPC may be common in the gastrointestinal tracts of children.
Characterization of Mycobacterium paratuberculosis and organisms of the Mycobacterium avium complex by restriction polymorphism of the rRNA gene region
Authors: Chiodini R J
Published: January 2020

Nineteen Mycobacterium paratuberculosis strains, including strains of bovine, caprine, ovine, cervid, subhuman primate, and human origins, were compared with organisms of the M. avium complex by restriction fragment length polymorphism with a 5S rRNA gene probe as the reference DNA. Mycobacterial DNA was extracted, digested with several restriction enzymes, subjected to electrophoresis and Southern blotting, and then hybridized with a 5S rRNA gene probe from Escherichia coli. Hybridizing bands were visualized by autoradiography, and the sizes of the resulting rRNA fragments in kilobases were determined. Base substitutions were calculated on the basis of the number of shared fragments between species and strains. It was determined that M. paratuberculosis and the M. avium complex possess a single copy of the rRNA genes within their genomes and that the M. avium complex and M. paratuberculosis are a group of closely related organisms, likely with a common ancestral link. In proximity to the 5S rRNA gene exists a region or regions which display polymorphisms that are capable of species and subspecies differentiation. M. paratuberculosis strains isolated from humans, subhuman primates, and animals were found to be genetically identical to each other. M. paratuberculosis strains lacked the genetic heterogeneity (restriction fragment length polymorphisms) characteristic of most species, suggesting that this organism has unidirectional genetic selection. It is therefore assumed to be biologically isolated, occupying a unique and specific biological niche. This homogeneity was present in all strains, including those of animal and primate (subhuman and human) origin and strains isolated from different parts of the world.
Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens
Authors: Shawar R M , Moore D G , LaRocco M T
Published: January 2020

Isolation of Nocardia spp. from clinical specimens can be enhanced by the use of paraffin baiting, which relies on the selective ability of the organism to metabolize paraffin. We evaluated 44 Nocardia isolates, 18 group IV mycobacterial isolates, and 4 Streptomyces isolates for growth on blood agar (BA) and on carbon-free agar containing single or double substrates as follows: paraffin agar (PA), gelatin agar (GA), urea agar (UA), PA-gelatin (PG), and PA-urea (PU). The growth rates of Nocardia spp. on BA, PA, PU, and PG were similar; but 3-day-old colonies were larger on BA for 20 (45%) isolates. After longer incubations (7 to 14 days), some Nocardia colonies were larger on PA, PG and PU than they were on BA. Despite variable morphologies on BA, colonies on PA, PG, and PU were consistently smooth, creamy, and raised. Compared with growth on BA, the growth of mycobacteria was much slower on PA, PG, and PU, with poor growth on UA and GA. The growth of Streptomyces spp. was greatly enhanced on GA, PG, UA, and PU and was poorest on PA. Twelve sputum specimens seeded with Nocardia asteroides (10(4) CFU/ml) were inoculated onto BA and all chemically defined media. Nocardiae were recovered from 6 to 12 specimens grown on BA, GA, and UA; 11 of 12 specimens grown on PG; and 12 of 12 specimens grown on PA and PU. Only PA was able to suppress the growth of other microorganisms that were present in sputum specimens. These results suggest that chemically defined media containing PA may be useful for the selective isolation of Nocardia spp. from contaminated clinical specimens.
Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis
Authors: Patel R J , Fries J W , Piessens W F , Wirth D F
Published: January 2020

Analysis of the 1,016-base-pair sequence of a putative probe for identification of Mycobacterium tuberculosis revealed two almost identical fragments of 507 and 509 bases. From this sequence two pairs of primers were synthesized (MtbAB and MtbCD), ranging from 18 to 22 nucleotides, for use in polymerase chain reactions (PCRs) with DNA from six reference strains of M. tuberculosis, as well as type strains of M. bovis, M. bovis BCG, M. kansasii, M. avium, M. intracellulare, and M. scrofulaceum. Although there was amplification of DNA from all mycobacterial strains included in the study, when used as probes, a predominant band, fragment CD from M. tuberculosis H37Rv DNA, proved to be more specific for strains of M. tuberculosis than the original probe, pMTb4, was. Amplified fragments from as little as 1 fg of DNA (equivalent to one-fifth of an organism) could be resolved on ethidium bromide-stained gels loaded with a 1/10 volume of PCR. Furthermore, it was possible to amplify specific DNA sequences from frozen M. tuberculosis H37Rv organisms which were thawed prior to PCR.
Detection of murine typhus infection in fleas by using the polymerase chain reaction
Authors: Webb L , Carl M , Malloy D C , Dasch G A , Azad A F
Published: January 2020

Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.
Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction
Authors: Pollard D R , Johnson W M , Lior H , Tyler S D , Rozee K R
Published: January 2020

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.
Clinical studies of a quadrivalent rotavirus vaccine in Venezuelan infants
Authors: Perez-Schael I , Blanco M , Vilar M , Garcia D , White L , Gonzalez R , Kapikian A Z , Flores J
Published: January 2020

Phase I studies of an oral quadrivalent rotavirus vaccine were conducted in 130 Venezuelan infants 10 to 20 weeks of age. The vaccine consists of a mixture of equal amounts of rhesus rotavirus (RRV) vaccine (serotype 3 [VP7]) and each of three human rotavirus-RRV reassortant strains: D x RRV (serotype 1 [VP7]), DS1 x RRV (serotype 2 [VP7]), and ST3 x RRV (serotype 4 [VP7]). Three different doses of the quadrivalent vaccine (0.25 x 10(4), 0.5 x 10(4), and 10(4) PFU of each component) were evaluated sequentially for safety and antigenicity in placebo-controlled, double-blind trials. Starting the day after vaccination, the infants were monitored by daily home visits for 7 days. Only minor reactions were observed during this period; these were limited to mild transient febrile episodes which began day 2 or 3 after vaccination and lasted 1 to 2 days in 15 to 30% of the infants. Serological studies demonstrated that 68 to 96% of the infants developed a rotavirus serum immunoglobulin A response following vaccination. However, when tested by plaque reduction neutralization assay against individual human rotavirus serotype 1, 2, 3, or 4, the response rates ranged from 4 to 23% with the low dose, 21 to 33% with the medium dose, and 32 to 58% with the high dose. Most (73 to 79%) infants developed neutralizing antibodies to RRV following administration of each dose schedule. Vaccine virus shedding was analyzed by utilizing tissue culture isolation of virus from stool. All of the infants who received the lower of medium dose and 89% of those fed the high dose shed one or more components of the vaccine. Analyses of rotavirus serotypes isolated from the stool of infants who received the 0.25 x 10(4) -PFU dose revealed that DS1 x RRV was the most commonly shed vaccine component, followed by RRV, D x RRV, and ST3 x RRV in that order.
Genotypical variation of Campylobacter pylori from gastric mucosa
Authors: Oudbier J H , Langenberg W , Rauws E A , Bruin-Mosch C
Published: January 2020

In a previous study, the recurrence of the Campylobacter pylori infection after apparently successful antibacterial therapy was determined to be due to recrudescence rather than reinfection. Although the DNA patterns of pre- and posttreatment isolates were very similar, we detected minor differences between the two patterns in about one third of the patients. These differences were not artifacts, but originated in the coexistence in the stomach of (sub)populations of bacteria with slightly different chromosomal DNAs, plasmids, or both. The presence of such (sub)populations was probably caused by mutation in vivo, as mutation in vitro was demonstrated in one patient after the original isolate was subcultured 10 times. Minor differences were not correlated with a difference in susceptibility to the antibiotic(s) that was used. An additional conclusion of this investigation was that the results of plasmid analysis should be interpreted very carefully when this method is used as an epidemiologic marker in the investigation of C. pylori infections.
Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry
Authors: Shankaran P , Reichstein E , Khosravi M J , Diamandis E P
Published: January 2020

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.
Human serum antibody response to the presence of Aeromonas spp. in the intestinal tract
Authors: Kuijper E J , van Alphen L , Peeters M F , Brenner D J
Published: January 2020

A bacterial agglutination assay, a toxin-neutralizing assay, and an enzyme-linked immunosorbent assay (ELISA) were used to compare antibodies against intestinal Aeromonas strains in serum samples from healthy carriers (n = 6), from patients with acute (n = 15) or chronic (n = 8) gastroenteritis, from patients with gastroenteritis caused by other enteropathogenic bacteria (n = 3), and from healthy blood donors (n = 50). Evaluation of the bacterial agglutination assay showed that it was not very useful. The sensitivity of the ELISA in patients with acute or chronic aeromonas-associated diarrhea was 30% (7 of 23 patients were positive), whereas the specificity was 74% (13 of 50 healthy donors were positive). Positive results in the ELISA correlated with immunoglobulin M and immunoglobulin G responses to lipopolysaccharides of homologous Aeromonas strains, as determined by gel immunoradioassay and Western immunoblot analysis. The sera showed cross-reactions with heterologous Aeromonas strains and with Escherichia coli strains. The toxin-neutralizing assay was positive in 5 of 11 patients who had developed acute severe diarrhea associated with cytotoxin-producing Aeromonas strains (46% sensitivity), whereas only 3 of 50 healthy donors had low serum titers of cytotoxin-neutralizing antibodies (94% specificity). All five patients were over 60 years of age. Cytotoxin-neutralizing activity was not observed in the sera of other groups of patients with aeromonads in their feces. We concluded that the three different serologic assays were not consonant with one another and that only the toxin-neutralizing assay distinguished patients with acute diarrhea from other groups of patients.
Immunologic characterization of a 35-kilodalton recombinant antigen of Mycobacterium tuberculosis
Authors: Rumschlag H S , Yakrus M A , Cohen M L , Glickman S E , Good R C
Published: January 2020

A 35-kilodalton (kDa) recombinant antigen (35-kDa antigen) produced by Escherichia coli JM107 carrying DNA from Mycobacterium tuberculosis was purified and immunologically examined by in vivo and in vitro methods. A monoclonal antibody (2B2) was produced against the 35-kDa antigen. The protein was purified from the insoluble fraction of the recombinant E. coli strain by either affinity chromatography with the 2B2 monoclonal antibody or preparative isoelectric focusing. In enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses, antibody to 2B2 reacted with whole-cell sonic extracts of M. tuberculosis and other slowly growing mycobacteria but not with two rapid growers, M. chelonae and M. fortuitum. An injection series totaling less than 1 mg of purified protein without adjuvant elicited a humoral response in guinea pigs. In one guinea pig, 10 micrograms of purified protein injected intradermally elicited both a humoral and a cell-mediated response. Results of these studies suggest that the 35-kDa antigen is a membrane-associated protein that stimulates both humoral and cell-mediated immune responses and should be evaluated as a vaccine candidate.
Use of live cold-adapted influenza A H1N1 and H3N2 virus vaccines in seropositive adults
Authors: Treanor J J , Roth F K , Betts R F
Published: January 2020

To investigate the immunogenicity and protective efficacy of cold-adapted influenza vaccine in individuals with underlying immunity to influenza A virus, we administered cold-adapted H1N1 and H3N2 vaccines to adults with prevaccination serum hemagglutination inhibition antibody titers of 1:16 or more and challenged them 1 month afterwards with homologous wild-type influenza A virus. Both cold-adapted vaccines were immunogenic in seropositive adults. In addition, individuals receiving cold-adapted vaccines had lower rates of virus shedding and illness following challenge with wild-type influenza virus than did unvaccinated seropositive volunteers.
Culture confirmation of Escherichia coli serotype O157:H7 by direct immunofluorescence
Authors: Tison D L
Published: January 2020

An evaluation of a fluorescein-labeled, polyclonal, affinity-purified goat antibody to Escherichia coli serotype O157:H7 (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) was conducted to determine the efficacy of this research reagent for the rapid direct immunofluorescence identification of E. coli O157:H7 isolated from fecal specimens cultured on sorbitol-MacConkey (SMAC) agar. The E. coli O157:H7 fluorescent-antibody conjugate proved to be 100% sensitive and specific for the rapid identification of non-sorbitol-fermenting E. coli O157:H7 from SMAC agar inoculated with fecal specimens. The addition of SMAC agar to the battery of primary isolation media routinely used for fecal specimens resulted in the identification of 16 patients with E. coli O157:H7 disease from a total of 799 fecal specimens cultured during 1988.
Rapid diagnosis by buffy coat smear of disseminated Mycobacterium avium complex infection in patients with acquired immunodeficiency syndrome
Authors: Nussbaum J M , Dealist C , Lewis W , Heseltine P N
Published: January 2020

A smear of the buffy coat of peripheral blood for acid-fast bacilli was assessed for sensitivity and specificity in the diagnosis of disseminated Mycobacterium avium complex (MAC) infection in acquired immunodeficiency syndrome (AIDS) patients. Seventeen AIDS patients with blood cultures positive for MAC had simultaneous quantitative blood cultures and buffy coat smears performed, as did 4 patients later proven not to have disseminated MAC. The sensitivity of the buffy coat smear for the detection of MAC was 35%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 22%. We conclude that the buffy coat smear is a rapid, simple, and specific method of diagnosis of disseminated MAC infection in AIDS patients, although it is not very sensitive.
Porcine rotaviruses antigenically related to human rotavirus serotypes 1 and 2
Authors: Bellinzoni R B , Mattion N M , Matson D O , Blackhall J , La Torre J L , Scodeller E A , Urasawa S , Taniguchi K , Estes M K
Published: January 2020

Fecal samples from rotavirus-infected piglets were characterized by a serotyping enzyme-linked immunosorbent assay (ELISA) by using monoclonal antibodies (MAbs) specific to human serotypes 1, 2, 3, and 4 (D. O. Matson, M. K. Estes, J. W. Burns, H. B. Greenberg, K. Taniguchi, and S. Urasawa, submitted for publication). Rotavirus in 19 of 25 specimens tested from two herds of pigs from Buenos Aires province, Argentina, were classified antigenically as follows: one serotype 1, four serotype 2, two serotype 3, and no serotype 4. Six specimens reacted with both serotype 1 and 2 MAbs, and viruses in six specimens probably belonged to other serotypes because they reacted only with a VP7 common epitope MAb. Two porcine rotavirus fecal samples found to contain both serotype 1 and 2 viruses by the MAb-based test and one found to contain a serotype 2 virus were grown in tissue culture. When plaque-purified preparations of these tissue culture-adapted viruses were analyzed in the serotyping ELISA, the C60 and C86 preparations reacted only as serotype 1 viruses, indicating that the original fecal samples, which showed multiple VP7 reactivities, were heterogeneous and apparently contained two types of viruses. Testing of plaque-purified C134 virus confirmed its serotype 2 reactivity. The MAb-based serotype designations of these viruses also were confirmed by using a neutralization immunoperoxidase focus reduction assay. This is the first report of the occurrence of serotype 1 and 2 rotaviruses in animals. The MAbs originally developed to serotype human rotaviruses can be utilized to type animal rotaviruses.
Sequential analysis of staphylococcal colonization of body surfaces of patients undergoing vascular surgery
Authors: Levy M F , Schmitt D D , Edmiston C E , Bandyk D F , Krepel C J , Seabrook G R , Towne J B
Published: January 2020

Slime-producing coagulase-negative staphylococci are pathogens in vascular surgery by virtue of their ability to adhere to and persist on prosthetic graft material. Inguinal and abdominal skin sites were cultured in 41 patients upon hospitalization, and slime production and antimicrobial susceptibility were assessed in all recovered staphylococcal isolates. Twenty-one patients eventually underwent lower-extremity revascularization. In the operative population, cultures were also obtained on the day of surgery and fifth postoperative day. All 21 patients received perioperative cefazolin. Of 327 coagulase-negative staphylococci recovered, Staphylococcus epidermidis (47%), S. haemolyticus (21%), and S. hominis (10%) were the predominant isolates. Slime-producing coagulase-negative staphylococci were recovered from 17 of 21 patients at admission but only from 8 of 21 patients on day 5 postoperation (P less than 0.05). S. epidermidis isolates demonstrated increasing multiple resistance from admission to 5 days postoperation to methicillin, gentamicin, clindamycin, erythromycin, and trimethoprim-sulfamethoxazole (P less than 0.05). All coagulase-negative staphylococcal isolates were susceptible to ciprofloxacin and vancomycin. Slime-producing capability was not associated with increased methicillin resistance for the recovered isolates. The data demonstrate that patients enter the hospital colonized with slime-producing strains of coagulase-negative staphylococci and that during hospitalization the staphylococcal skin burden shifts from a predominately susceptible to a resistant microbial population, which may enhance the importance of slime production as a risk factor in lower-extremity revascularization.
Phenotypic variation of Staphylococcus epidermidis in infection of transvenous endocardial pacemaker electrodes
Authors: Baddour L M , Barker L P , Christensen G D , Parisi J T , Simpson W A
Published: January 2020

Coagulase-negative staphylococci isolated from a patient with a pacemaker electrode infection were extensively evaluated by phenotypic and genotypic characterization. Findings from this evaluation were striking because different colony morphologic subtypes were recovered from blood and resected pacemaker electrodes. Staphylococci from each colony subtype (LBL, LBV, LBP, LBS) were identified as slime-producing strains of Staphylococcus epidermidis sensu stricto. Direct plating of isolates from a restricted electrode revealed a mixture of colony phenotypes when examined on a high-salt, low-glucose medium, Memphis agar. Bacteriophage typing employing 17 different phages and plasmid profile analysis were largely unsuccessful in further characterizing bacterial cells of each of the four colony morphotypes. On the other hand, restriction endonuclease analysis by EcoRI digestion of the chromosomal DNA demonstrated the probable common clonal origin of the four colony phenotypes.
Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes
Authors: Bracha R , Diamond L S , Ackers J P , Burchard G D , Mirelman D
Published: January 2020

Most individuals infected with Entamoeba histolytica are reported to be clinically asymptomatic. On the basis of the electrophoretic migration of hexokinase and phosphoglucomutase isoenzymes, two groups of E. histolytica isolates have been classified. Those derived from symptomatic cases were found to have fast-migrating hexokinase bands and were labeled pathogenic. The others, isolated from cyst passers, had (in most cases) slow-migrating bands and were called nonpathogenic. Differences between these two groups of E. histolytica were found recently at the DNA level. Two sets of different DNA probes derived from tandemly repeated sequences present in extrachromosomal circular DNA elements in each group of E. histolytica were characterized. Using these probes with procedures for direct hybridization of trophozoites on nylon membranes, we could correctly correlate hexokinase electromobility with the DNA hybridization signal of 81 different isolates of E. histolytica. The advantages of using DNA probes lie in their sensitivity (fewer than 200 trophozoites can be detected) and specificity. The probes hybridized only with amebae from the E. histolytica species and not with other enteric protozoa and can be useful as a diagnostic tool.
Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli
Authors: De Rycke J , González E A , Blanco J , Oswald E , Blanco M , Boivin R
Published: January 2020

We have characterized the in vitro and in vivo toxic properties of cell sonic extracts from 22 animal and human clinical isolates of Escherichia coli that caused both necrosis in the rabbit skin and multinucleation in tissue cultures, two toxic properties previously reported as being specific for E. coli cytotoxic necrotizing factor (CNF). Two distinct toxic phenotypes were observed. Type 1, which was displayed by originally described CNF strains, was characterized by extensive multinucleation and rounding of cells in HeLa cell culture assays, moderate necrosis in the rabbit skin test, and absence of necrosis in the mouse footpad test. Type 2, which has recently been shown to be associated with E. coli Vir plasmid, was characterized by moderate multinucleation, by polymorphism and elongation of HeLa cells, and by an intense necrotic response in both the rabbit skin test and the mouse footpad test. The distinction between the two cytotoxins accounting for these effects (CNF 1 and CNF 2), together with their partial relatedness, was confirmed by seroneutralization studies of both cytopathic effects and necrosis in the rabbit skin test. In addition, type 2 extracts were more lethal in the mouse intraperitoneal test and induced a moderate, although not totally repetitive, fluid accumulation in the ileal loop test. The original toxic properties of these recently recognized categories of E. coli strains, together with their association with enteritis and septicemia, suggest that these strains may play a significant role in pathology.
DNA and protein analyses of tick-derived isolates of Borrelia burgdorferi from California
Authors: LeFebvre R B , Lane R S , Perng G C , Brown J A , Johnson R C
Published: January 2020

Nine isolates of Borrelia burgdorferi from ixodid ticks collected in northern California were characterized. Restriction endonuclease analysis, pulsed-field gel electrophoresis, and Western blot (immunoblot) analysis were used in this study. Four isolates were very similar to each other. The others shared some similarities but were classified as having unique genotypes. A strain from an Ixodes neotomae tick displayed the greatest genetic and antigenic diversity when compared to the isolates collected from Ixodes pacificus ticks. A computerized library based on DNA banding patterns of the isolates by restriction enzyme analysis is also reported. This library was created by using a scanning laser densitometer.
Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures
Authors: Schöningh R , Verstijnen C P , Kuijper S , Kolk A H
Published: January 2020

A simple enzyme-linked immunosorbent assay was developed for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii. Six monoclonal antibodies were used: two (F23-49 and F24-2) were specific for the M. tuberculosis complex, two (F85-2 and F85-10) were specific for the identification of the M. avium complex, one (F126-22) was specific for M. kansasii, and one (F141-3) was broadly reactive and distinguished mycobacteria from other bacteria. In the enzyme-linked immunosorbent assay only 10(6) to 10(7) bacteria derived from early cultures (2 to 3 weeks) were needed for each monoclonal antibody. For the M. tuberculosis complex the sensitivity and specificity were both 100%. For the M. avium complex the specificity was 100% and the sensitivity was 70%. The three M. kansasii strains tested could all be identified as M. kansasii.
Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry
Authors: McSharry J J , Costantino R , Robbiano E , Echols R , Stevens R , Lehman J M
Published: January 2020

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.
Rapid detection of respiratory syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper
Authors: Jalowayski A A , Walpita P , Puryear B A , Connor J D
Published: January 2020

We compared the Rhinoprobe scraping technique for collection of superficial nasal mucosa epithelial cells and rapid detection of respiratory syncytial virus by immunofluorescence with paired, swab-collected specimens for virus culture from 1,257 infants and children with acute respiratory infections. Compared with viral culture as the reference test, the sensitivity, specificity, and accuracy of the immunofluorescence test were 83.6, 93.6, and 91.3%, respectively. We found the Rhinoprobe method safe, easy to use, and helpful in obtaining large quantities of epithelial cells for detection of respiratory syncytial virus and other respiratory viruses.
Sandwich enzyme immunoassay and latex agglutination test for herpes simplex virus keratitis
Authors: Lee S F , Pepose J S
Published: January 2020

We determined the in vitro sensitivities and the diagnostic efficacies of a commercially available latex agglutination test and an enzyme-linked immunosorbent assay using a rabbit model of herpes simplex virus type 1 dendritic keratitis. In contrast to the latex agglutination test, the HERPCHEK enzyme-linked immunosorbent assay offers an extremely sensitive and reliable means of laboratory diagnosis of experimental herpes simplex virus keratitis and is more rapid than viral isolation in tissue culture.
Comparison of three assay systems for detection of enterotoxigenic Escherichia coli heat-stable enterotoxin
Authors: Cryan B
Published: January 2020

In this study, a commercial DNA-DNA hybridization kit for the detection of Escherichia coli heat-stable enterotoxin is compared with a competitive enzyme-linked immunosorbent assay (ELISA) and the suckling mouse bioassay. Taking the suckling mouse assay as the "gold standard," the gene probe was the more specific and the ELISA was the more sensitive of the assays. The ELISA and the suckling mouse test are semiquantitative. The ELISA was the most rapid method, most amenable to automation, and most suitable for the examination of large numbers of specimens. The gene probe is particularly applicable in relatively primitive laboratory conditions. The suckling mouse assay was the least suitable system for the examination of large numbers of specimens.
Urease-negative Nocardia asteroides causing cutaneous nocardiosis
Authors: Boiron P , Lafaurie C , Rabbache A , Brown J , Carteret R , Petit J
Published: January 2020

We report a case of cutaneous nocardiosis, caused by a urease-negative strain of Nocardia asteroides, in a patient who had undergone long-term corticosteroid therapy. The microbiological characteristics of the isolate were typical of N. asteroides except for the failure of the isolate to hydrolyze urea. During the course of routine identification, laboratory specialists should be aware of the occasional occurrence of atypical strains of N. asteroides.
Temperature range for growth of Escherichia coli serotype O157:H7 and selected coliforms in E. coli medium
Authors: Raghubeer E V , Matches J R
Published: January 2020

Escherichia coli serotype O157H:7 and five other fecal and nonfecal coliforms were tested for minimum and maximum temperatures for growth in E. coli medium by using a temperature gradient incubator with a mean temperature increment of 1.67 degrees C (+/- 0.392). The temperature range for growth of E. coli O157:H7 is inconsistent with that of other fecal coliforms, suggesting that this pathogen is excluded with standard enumeration procedures used for foods and water.
Evaluation of the Recombigen HIV-1 Latex Agglutination Test
Authors: Starkey C A , Yen-Lieberman B , Proffitt M R
Published: January 2020

The Recombigen HIV-1 Latex Agglutination (LA) Test was recently licensed by the U.S. Food and Drug Administration for use as a rapid screening assay for human immunodeficiency virus type 1 (HIV-1) antibodies. However, its performance in various settings and in different populations has not been firmly established. Consequently, we evaluated the test in the Cleveland Clinic Retrovirus Laboratory, a regional reference laboratory for HIV diagnostic testing and a testing laboratory for the Ohio Department of Health Anonymous HIV Testing and Counseling Program. Serum samples from 93 individuals presumed to be at high risk for HIV infection were evaluated. The sera were initially tested for HIV antibodies by enzyme-linked immunosorbent assay (ELISA). All repeatedly reactive sera were subjected to confirmatory Western blot (WB; immunoblot) testing. Of 97 serum specimens tested (5 were from one seroconverter), 44 were repeatedly reactive by ELISA and 53 were nonreactive. Of the reactive serum specimens, 31 were confirmed positive and 12 were indeterminate by WB. All of the sera were coded and then retested by the LA test. Of 53 serum specimens nonreactive by ELISA, 51 were also nonreactive in the LA test. Of the 44 serum specimens reactive by ELISA, 16 were nonreactive by LA; however, 3 of the latter were WB positive. No serum specimen with an ELISA ratio (specimen optical density/cutoff optical density) of less than 2.1 scored reactive in the LA test. The LA test was positive for only two of five consecutive serum specimens from a seroconverter despite the fact that all but the earliest of these were ELISA reactive and WB positive. Although the LA test appears to be an adequate first-line screening test when appropriately used according to the directions of the manufacturer, our data suggest that occasional sera with low levels of reactivity by ELISA may not be readily detected as reactive by the LA test.
Distinct populations of rotaviruses circulating among neonates and older infants
Authors: Tam J S , Zheng B J , Lo S K , Yeung C Y , Lo M , Ng M H
Published: January 2020

We obtained three stool specimens from each of 371 neonates. Two specimens were obtained between days 1 and 3 after birth, while they were in the hospital, and one specimen was obtained between days 6 and 14 after birth, after they had been discharged from the hospital. Seventy neonates excreted human rotavirus (HRV) while they were in the hospital, and the incidence rate for the cohort was 0.094 episodes per infant day. The incidence rate of community-acquired neonatal infections was markedly reduced to 0.022 episodes per infant day, with eight additional episodes of infection being detected after the infants were discharged from the hospital. Nevertheless, this was higher than the incidence of community-acquired HRV infection of 0.0037 episodes per infant day previously estimated by serology for the same cohort during the subsequent 2 years of infancy. None of the 78 episodes of neonatal HRV infection was accompanied by diarrhea. There were at least 44 distinct electropherotypes of HRV circulating among older infants in the community during the study period, and they comprised at least four different serotypes. Despite the genetic and antigenic diversity of the prevalent HRV isolates, only five electropherotypes with either serotype 2 or 4 specificity were isolated from the neonates, while serotype 1 and 3 viruses were not detected. Two of these electropherotypes, including one which was isolated from 57 of the 78 infants with episodes of infection, were isolated exclusively from the neonates. The other three electropherotypes were also isolated from the older infants; one was a major electropherotype and two were minor electropherotypes which were prevalent among the older infants. These results suggest that distinct populations of HRV cocirculate among neonates and older infants.
Septicemia caused by cysteine-dependent Escherichia coli
Authors: Yuen K Y , Seto W H , Tsui K H , Hui W T
Published: January 2020

A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteine-dependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.
Presumptive identification of enteroviruses with RD, HEp-2, and RMK cell lines
Authors: Johnston S L , Siegel C S
Published: January 2020

The limited supply of the Lim Benyesh-Melnick antiserum pools for the typing of enteroviruses has made this test inappropriate for routine use in most clinical laboratories. We studied the correlation between the enterovirus groups and the cell lines on which they displayed cytopathic effect in order to make identifications without using the neutralization test. This study indicated that a presumptive identification of the enterovirus group could be made on the basis of characteristic cytopathic effect displayed after passage into rhabdomyosarcoma (RD), HEp-2, and primary rhesus monkey kidney (RMK) cells. Echoviruses and group A coxsackieviruses could be isolated in RD and RMK cells but not HEp-2 cells. Group B coxsackievirus could be isolated in RMK and HEp-2 cells but not RD cells. Poliovirus could be isolated in all three cell lines. We recommend the use of these cell lines to make presumptive enterovirus group identifications for routine viral isolates.
Enzymatic activity profiling as a potential biotyping method for Ajellomyces dermatitidis
Authors: Summerbell R C , Kane J , Pincus D H
Published: January 2020

Enzyme profiling was investigated as a means of recognizing biotypes and individual strains of Ajellomyces dermatitidis (anamorph, Blastomyces dermatitidis). Eighteen North American and 2 African representatives were tested with the Yeast-IDENT enzymatic activity profiling system (Analytab Products, Plainview, N.Y.). Significant variation was found between isolates, particularly in beta-galactosaminidase activity.
Detection of Candida antigenuria in disseminated candidiasis by immunoblotting
Authors: Ferreira R P , Yu B , Niki Y , Armstrong D
Published: January 2020

Immunoblotting (Western blotting) was used to detect Candida albicans antigens in urine of 10 patients with disseminated candidiasis who had two or more positive blood cultures. Twelve urine samples were examined; and antigenuria was found in five of six patients with C. albicans infections, in one patient with a mixed Candida infection (including C. albicans), and in one of two patients with C. tropicalis infection. All except one specimen was collected from 2 to 12 days after initiation of amphotericin B therapy. Positive samples showed different numbers of bands in Western blots with an antigen that had an apparent molecular weight of 47,000 in common. This antigen was not found in the urine of patients who had more than 5 days of therapy for candidiasis and who were responding to therapy. The results suggest that Western blotting for C. albicans antigens in urine may be a useful method for the diagnosis of disseminated candidiasis and for evaluating antifungal treatment.
Rises in antibody to human herpesvirus 6 detected by enzyme immunoassay in transplant recipients with primary cytomegalovirus infection
Authors: Chou S W , Scott K M
Published: January 2020

Immunoglobulin G to human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) in sera from solid organ recipients was measured by an enzyme-linked immunoassay (ELISA) before and after transplant. The HHV-6 ELISA was developed from glycine extracts of HHV-6-infected and uninfected HSB-2 cells. At a serum dilution of 1:500, 80 (91%) of 88 recipients were seropositive for HHV-6 before transplant, while only 14 (16%) were seropositive for CMV. Posttransplant HHV-6 serologic rises were observed in 38 (43%) recipients; rises in 25 of these recipients were associated with primary CMV infection. Titration of sera revealed much higher HHV-6 titer rises among those with primary CMV infection than among those with CMV reactivation or with no CMV infection. Elevated HHV-6 antibody titers persisted for up to 2 years after primary CMV infection. No correlation was noted between CMV and HHV-6 antibody titers in individual serum samples.
Rabies diagnostic reagents prepared from a rabies N gene recombinant expressed in baculovirus
Authors: Reid-Sanden F L , Sumner J W , Smith J S , Fekadu M , Shaddock J H , Bellini W J
Published: January 2020

A gene encoding the nucleoprotein (N) of rabies virus was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant gene expression was controlled by the strong polyhedrin gene promoter. Insect cells (Spodoptera frugiperda) infected by a baculovirus recombinant containing the rabies virus N gene produced abundant amounts of a novel 55-kilodalton protein of a size comparable to that of the rabies virus N protein, as demonstrated by polyacrylamide gel electrophoresis. This new gene product possessed the antigenic and immunogenic properties of native viral N protein, as shown by the ability of the new protein to react in immunoprecipitation and immunofluorescence assays with antirabies antibodies, to serve as a substitute for infectious rabies virus in adsorbing suspensions for diagnostic tests, and to induce high-titered antiserum. The baculovirus expression system provides a safe, convenient, and inexpensive source of rabies virus N protein for the production of both antiserum and adsorbing suspensions for use in rabies diagnoses.