bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of serum antibody responses to natural rotavirus infections in children by VP7-specific epitope-blocking assays
Authors: Matson D O , O'Ryan M L , Pickering L K , Chiba S , Nakata S , Raj P , Estes M K
Published: January 2020

Knowledge of the immune response to rotavirus is crucial for vaccine development. We compared an epitope-blocking assay (EBA) that uses VP7-specific monoclonal antibodies with neutralization assays (NAs) with polyclonal antisera for detecting serum antibody responses after natural rotavirus infection in children. Twenty-six serum pairs from children living in an orphanage with and without symptoms during two rotavirus outbreaks were evaluated for VP7 type 1-, 2-, 3-, and 4-specific antibody responses. In the first outbreak, which was caused by a VP7 type 3 strain, homotypic antibody responses were detected in 11 of 11 symptomatic children by NA and in 10 of 11 symptomatic children by EBA. Heterotypic antibody responses were detected more frequently (12 of 15 children) by NA than by EBA, and the heterotypic epitope-blocking antibody responses occurred in children older than 14 months of age. Antibody responses in asymptomatic children were more commonly detected by EBA than by NA. EBA results from the sera of children in the second outbreak indicated that it was caused by VP7 type 4, whereas NA results suggested it was caused by VP7 type 3. Our results confirm that EBA is a sensitive and specific method for determining VP7 type-specific immune responses after natural rotavirus infections.
Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens
Authors: Ryan-Poirier K A , Katz J M , Webster R G , Kawaoka Y
Published: January 2020

Directigen FLU-A, a new enzyme immunoassay membrane test, rapidly detects influenza A virus antigen in specimens from patients. Nasopharyngeal washes and pharyngeal gargles were used to determine the effectiveness of the assay as applied to different types of routinely collected clinical samples. All specimens had been previously shown to contain influenza A virus by virus isolation in tissue culture. Directigen FLU-A was 90% sensitive (95% confidence interval, 56 to 99.7%) with nasopharyngeal washes but only 39% sensitive (95% confidence interval, 17 to 64%) with pharyngeal gargles (P = 0.018) when used with samples containing similar amounts of infectious virus (50% tissue culture infective dose, 1.0 to 4.5). The intensity of the positive reaction with Directigen FLU-A did not correlate with the amount of virus in the specimens. Directigen FLU-A was found to detect cell-associated antigen more readily than free virus; only 20 infected cells were required to identify cell-associated influenza A virus antigen, whereas the limit of detection for free virus was 1.63 x 10(3) infectious virus particles. These findings suggest that Directigen FLU-A detects the cell-associated antigen present in clinical specimens rather than free virus. In addition, Directigen FLU-A detected avian and swine influenza A viruses in both cloacal swabs (75% sensitivity) and swine lung homogenates (86% sensitivity), indicating its potential usefulness in the surveillance of nonhuman influenza A viruses.
Clinical evaluation of oral fluid samples for diagnosis of viral hepatitis
Authors: Thieme T , Yoshihara P , Piacentini S , Beller M
Published: January 2020

Oral fluid samples were compared with serum samples as a specimen source for hepatitis A, B, and C virus markers. Oral fluid was obtained with a treated absorbent pad and tested by using existing commercial enzyme immunoassays with only minor modifications. Compared with serum sampling the sensitivity and specificity of oral sampling were 100% (51 of 51 samples) and 98% (46 of 47 samples) for hepatitis A virus immunoglobulin M, 100% (29 of 29 samples) and 100% (29 of 29 samples) for hepatitis B virus surface antigen, and 100% (13 of 13 samples) and 100% (13 of 13 samples) for hepatitis C virus antibody, respectively. The decline of hepatitis A virus immunoglobulin M in oral samples was parallel to, though somewhat more rapid than, that of hepatitis A virus immunoglobulin M in serum samples. It is proposed that oral sampling represents a safer and more convenient procedure for reliable hepatitis virus testing than blood sampling and that it has wide application in patient and outbreak management.
Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A
Authors: De Girolami P C , Hanff P A , Eichelberger K , Longhi L , Teresa H , Pratt J , Cheng A , Letourneau J M , Thorne G M
Published: January 2020

The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study. Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells. The tubes and plates were examined at 24 and 48 h for cytotoxicity. Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C. difficile were used to evaluate discrepant results. Of 504 samples, 66 were positive and 409 were negative by both tests. Eight samples had indeterminate PTA EIA results and were excluded from this analysis. Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive. Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC. The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA. The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA. We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B.
Use of a dispersed repetitive DNA element to distinguish clinical isolates of Cryptococcus neoformans
Authors: Spitzer E D , Spitzer S G
Published: January 2020

We isolated a recombinant phage from a Cryptococcus neoformans genomic library that contains a member of a dispersed family of repetitive DNA elements. This clone, CNRE-1, hybridized to at least seven chromosomes in C. neoformans on the basis of pulsed-field gel analysis. Hybridization of CNRE-1 to restriction digests of genomic DNA confirmed that there are multiple copies of this element and that restriction fragment length polymorphisms are present in strains from different serotypes of C. neoformans. The utility of this probe as an epidemiologic marker was determined by testing cryptococcal isolates from a single hospital. Five isolates from four patients were closely related to a serotype A reference strain, whereas five other isolates from four additional patients exhibited distinct patterns. In two patients, the isolates obtained during recurrent cryptococcal infections were identical to the original isolates.
Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp
Authors: Kaltenboeck B , Kousoulas K G , Storz J
Published: January 2020

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.
Evaluation of four serological tests for bovine paratuberculosis
Authors: Sockett D C , Conrad T A , Thomas C B , Collins M T
Published: January 2020

The standard complement fixation (CF) test, a commercial agarose gel immunodiffusion (AGID) test (ImmuCell Corporation, Portland, Maine), and two commercial enzyme-linked immunosorbent assays (ELISAs; Allied Laboratories, Glenwood Springs, Colo. [Allied ELISA], and the CSL, Limited, [Parkville, Victoria, Australia] enzyme immunoassay [CSL ELISA]) were evaluated by using sera from the Repository for Paratuberculosis Specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of dairy cattle without diarrhea or weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. None of the cattle evaluated had been vaccinated for paratuberculosis. Among 177 M. paratuberculosis-infected cattle, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA had test sensitivities of 38.4, 26.6, 58.8, and 43.4%, respectively, and specificities of 99.0, 100.0, 95.4, and 99.0%, respectively. Only 108 of the infected cattle were confirmed by culture or by a commercial DNA probe (IDEXX Corporation, Portland, Maine) to be shedding the organism in fecal samples at the time of serological testing. Among the 108 M. paratuberculosis fecal shedders, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA were positive for 54.6, 40.7, 65.7, and 56.5% of the cows, respectively; and among the 69 cows that were nonshedders, the tests were positive for 14.5, 4.3, 47.8, and 24.6% of the cows, respectively. There was a significant difference (P < 0.05) in the rate of positive test results for all four tests between these two groups of animals. The CF test performed well when it was compared with the other serological tests if a titer of >/- 1.8 was classified as a positive test result.
Isolation and purification of two immunodominant antigens from Nocardia brasiliensis
Authors: Vera-Cabrera L , Salinas-Carmona M C , Welsh O , Rodriguez M A
Published: January 2020

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.
Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction
Authors: Le Bouguenec C , Archambaud M , Labigne A
Published: January 2020

Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.
Detection of bacterial growth by gas absorption
Authors: Waters J R
Published: January 2020

When 24 different aerobic organisms were grown in a shaken culture, all were found to first absorb gas from the headspace. In a rudimentary medium, such as tryptic soy broth, 16 of the 24 organisms did not produce gas following the initial gas absorption. We have developed a simple, noninvasive method for detecting both gas absorption and production in multiple culture vials. The time to positivity was compared with that obtained by the BACTEC 460 blood culture system. For nearly all of these organisms, there was no difference. For some of those organisms that did not produce gas, e.g. Staphylococcus epidermidis, Moraxella osloensis, and Neisseria meningitidis, detection by gas absorption was a few hours faster. Gas absorption appears to be a promising technique for a new automated blood culture system because of its simplicity and because medium without special additives can be used to detect organisms that do not produce gas.
Ribosomal DNA probe for differentiation of Babesia microti and B. gibsoni isolates
Authors: Conrad P A , Thomford J W , Marsh A , Telford S R , Anderson J F , Spielman A , Sabin E A , Yamane I , Persing D H
Published: January 2020

The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B. microti DNA using universal primers directed against highly conserved portions of the eukaryotic 16S-like rRNA gene. The chemiluminescent rDNA probe hybridized to Southern blots of restriction endonuclease-digested DNA preparations of different isolates of B. gibsoni from infected dogs and B. microti from infected humans and white-footed mice. Restriction fragment length polymorphisms served to differentiate these species. Although the hybridization patterns seen with DNAs from six B. microti isolates did not vary, those of the five B. gibsoni isolates did indicate genotypic variation. We concluded that isolates of B. microti and B. gibsoni can be differentiated on the basis of restriction fragment length polymorphism detected with a chemiluminescent rDNA probe.
Mycobactin analysis as an aid for the identification of Mycobacterium fortuitum and Mycobacterium chelonae subspecies
Authors: Bosne S , Lévy-Frébault V V
Published: January 2020

Mycobactin patterns from 65 Mycobacterium fortuitum and Mycobacterium chelonae strains have been determined by thin-layer chromatography. By use of a rich liquid medium containing an iron chelator (ethylenediamine-di-o-hydroxyphenylacetic acid [EDDA]) to ensure iron starvation, all strains were able to form mycobactins. The method developed here allows sensitive detection of mycobactin by thin-layer chromatography from as little as 5 ml of culture after a 2-week incubation. Within M. fortuitum two mycobactin patterns were identified, whereas within M. chelonae four were recognized. Comparisons with the subspecific identification performed by using biochemical tests showed that 73% of the M. fortuitum subsp. fortuitum strains shared the same mycobactin pattern (designated F), whereas 75% of the M. fortuitum subsp. peregrinum strains shared the other mycobactin pattern (designated P). Within the M. fortuitum strains that cannot be assigned to a subspecies on the basis of their biochemical features, only F and P patterns were determined. Similarly, 93% of the M. chelonae subsp. chelonae strains produced the so-called C1 and C2 patterns and 86% of the M. chelonae subsp. abscessus strains produced A1 and A2 patterns. C2 and A2 were the patterns most frequently encountered; they were represented by 65 and 50% of the M. chelonae subsp. chelonae and M. chelonae subsp. abscessus strains, respectively. Within the biochemically M. chelonae strains that did not fit any subspecies on the basis of biochemical test results, C1, C2, and A1 patterns were found. Whereas about 30% of both M. fortuitum and M. chelonae strains cannot be characterized to the subspecies level on the basis of biochemical tests, 100% of the strains of both species can be characterized on the basis of mycobactin patterns.
Repetitive sequences cloned from Leptospira interrogans serovar hardjo genotype hardjoprajitno and their application to serovar identification
Authors: Pacciarini M L , Savio M L , Tagliabue S , Rossi C
Published: January 2020

We selected, from a genomic library of Leptospira interrogans serovar hardjo genotype hardjoprajitno, two probes containing repetitive sequences (pL1 and pL590). The hybridization patterns of these probes to DNA isolated from a variety of Leptospira serovars were examined and their ability to detect subtle differences at the genomic organization level was established. We identified the DNA fragments within pL1 and pL590 which are sufficient to yield polymorphic hybridization patterns; these results define the upper size limit of two novel repetitive elements in the Leptospira genome. The pattern and degree of hybridization observed for the serovars tested in this work were used to divide Leptospira spp. into groups which share genetic relatedness; our conclusions are consistent with previous classifications by other authors.
Identification of Aeromonas strains to the genospecies level in the clinical laboratory
Authors: Abbott S L , Cheung W K , Kroske-Bystrom S , Malekzadeh T , Janda J M
Published: January 2020

One hundred thirty-three strains of Aeromonas (human, n = 102; animal, n = 16; environmental, n = 15) previously identified to the DNA group level by molecular methods were biochemically analyzed for 58 properties. On the basis of the use of between 9 and 16 selected tests, 132 of the 133 strains (99%) could be assigned to their correct hybridization group using this biochemical scheme. The results suggest a feasible approach for identifying aeromonads to genospecies level under appropriate conditions.
Evaluation of the 4-hour RapID NF Plus method for identification of 345 gram-negative nonfermentative rods
Authors: Kitch T T , Jacobs M R , Appelbaum P C
Published: January 2020

The ability of the RapID NF Plus system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to identify 345 nonfermentative gram-negative rods was evaluated. Kits were inoculated with no. 1 McFarland suspensions, and reactions were interpreted after a 4-h incubation at 35 degrees C. Overall, the method correctly identified 311 strains (90.1%) without additional tests and 21 strains (6.1%) with additional tests, and 13 strains (3.8%) were misidentified. Five of 13 misidentified strains were Alcaligenes faecalis-Alcaligenes odorans misidentified as Alcaligenes xylosoxidans; however, all strains were xylose negative but nitrate positive and could have been A. faecalis group I-Alcaligenes piechaudii. The system does not differentiate between Pseudomonas fluorescens and Pseudomonas putida, and all Acinetobacter species are identified as Acetinobacter calcoaceticus. Additionally, no subspecies differentiation is made between A. xylosoxidans subsp. xylosoxidans and A. xylosoxidans subsp. denitrificans. All strains of the former Flavobacterium group IIb are identified as Flavobacterium indologenes-Flavobacterium gleum, and no species identification of the genus Methylobacterium is attempted. The system is easy to set up and interpret and provides an accurate commercial nonautomated method for same-day identification of gram-negative nonfermenters.
MIC and disk diffusion quality control guidelines for Neisseria gonorrhoeae susceptibility tests of cefdinir, cefetamet, CI-960, fleroxacin, lomefloxacin, and temafloxacin
Authors: Erwin M E , Jones R N , Koontz F P , Gerlach E H , Murray P R , Washington J A
Published: January 2020

Cefdinir (FK482), cefetamet (Ro 15-8074), CI-960, fleroxacin, lomefloxacin, and temafloxacin have potent activities against Neisseria gonorrhoeae. They were tested in a multilaboratory study to establish quality control guidelines. Quality control ranges for N. gonorrhoeae ATCC 49226 were determined by using multiple GC agar lots, three disk lots, and a number of test replicates consistent with the M23-T guidelines of the National Committee for Clinical Laboratory Standards. The MIC ranges included 2 to 4 log2 dilution steps. The recommended inhibition zone diameter ranges were generally 7 to 8 mm and included greater than or equal to 91.3% of all recorded study results.
Isolation of Nocardia asteroides from respiratory specimens by using selective buffered charcoal-yeast extract agar
Authors: Kerr E , Snell H , Black B L , Storey M , Colby W D
Published: January 2020

Nocardiosis is difficult to diagnose clinically and by laboratory methods. A patient presented with disease compatible with pulmonary malignancy, but Nocardia asteroides was isolated on buffered charcoal-yeast extract agar. Investigation revealed that this medium may be a suitable selective primary isolation medium for Nocardia species from respiratory specimens.
Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids
Authors: Floyd M M , Silcox V A , Jones W D , Butler W R , Kilburn J O
Published: January 2020

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.
Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction
Authors: Buck G E , O'Hara L C , Summersgill J T
Published: January 2020

Several simplified methods for treating mycobacteria to release DNA for amplification by the polymerase chain reaction (PCR) were investigated. The most effective of the methods was sonication. Samples were placed in screw-capped microcentrifuge tubes that were then placed in a plastic rack. The rack was floated in a dish of water next to the ultrasonic probe so that the ultrasonic energy was transmitted through the walls of the tubes. This allowed multiple samples to be processed safely and effectively. Forty-three clinical samples were processed by this procedure, and the crude preparations were analyzed for Mycobacterium tuberculosis by PCR. Twenty-six of these specimens contained M. tuberculosis, and 17 either had no growth or contained other species of mycobacteria. Twenty-four of the 26 (92%) positive specimens were correctly identified, and all of the negative specimens were correctly identified. This sonication procedure appears promising as a rapid, simple means of treating clinical specimens containing mycobacteria for PCR analysis.
Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis
Authors: Broitman N L , Floyd C M , Johnson C A , de la Maza L M , Peterson E M
Published: January 2020

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.
New plate medium for growth and detection of urease activity of Helicobacter pylori
Authors: Cellini L , Allocati N , Piccolomini R , Di Campli E , Dainelli B
Published: January 2020

A new medium for detection of urease activity and isolation of Helicobacter pylori is proposed. This medium, containing Columbia Agar Base, was supplemented with IsoVitaleX, hemin, urea, and phenol red (nonselective medium [NSM]). Both bacterial growth and color change were evaluated and compared with growth in the same medium supplemented with cefsulodin, vancomycin, polymyxin B sulfate, and amphotericin B (selective medium [SM]). Twenty-five recent clinical isolates and antral biopsy specimens from 33 patients who underwent endoscopy were examined. The isolates showed a rapid color change and good growth at 5 days of incubation with NSM and SM. H. pylori-positive biopsies revealed a color change within 36 h, and bacterial growth was better appreciated in NSM, but with more contaminating flora than in SM.
Identification of group A rotavirus gene 4 types by polymerase chain reaction
Authors: Gentsch J R , Glass R I , Woods P , Gouvea V , Gorziglia M , Flores J , Das B K , Bhan M K
Published: January 2020

Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
Detection of antibodies inhibiting the ADP-ribosyltransferase activity of pertussis toxin in human serum
Authors: Kaslow H R , Platler B W , Blumberg D A , Cherry J D
Published: January 2020

Bordetella pertussis produces a protein virulence factor termed pertussis toxin. Many candidate pertussis vaccines are based on the rationale that an immune response that neutralizes the virulence activities of this toxin, which are thought to arise from its catalytic ADP-ribosyltransferase activity, would be beneficial. The report describes two methods that quantify the inhibition of this activity by human serum. One, termed a direct assay, involves an initial incubation of toxin with serum, a second incubation that activates the toxin, and a third incubation that measures the ADP-ribosyltransferase activity of the mixture. The other assay, termed a plate assay, involves immobilization of the toxin, exposure of the immobilized toxin to serum and washing of the plate, and then activation and assay of the toxin's ADP-ribosyltransferase activity. The plate assay may be more selective than the direct assay in terms of identifying antibodies that neutralize the toxin in vivo. Sera from controls, selected patients presenting with cough, and vaccinated infants were first analyzed by the direct assay. In contrast to sera from controls, sera from several of the patients and vaccinated infants strongly inhibited activity. Dose-response curves of inhibition were determined for samples from three vaccinated infants by both the direct and plate assays. One of the samples had a dose-response curve of a different shape and thus differed not only in titer but also in functional characteristics. A comparison of inhibition of ADP-ribosyltransferase activity and neutralization in a CHO cell assay indicated that there was incomplete agreement between the two assays. Taken together, these results indicate that measurement of inhibition of ADP-ribosyltransferase activity by human serum is practical and may be useful in the evaluation of responses to pertussis vaccines.
Humoral immune responses to VP4 and its cleavage products VP5* and VP8* in infants vaccinated with rhesus rotavirus
Authors: Padilla-Noriega L , Fiore L , Rennels M B , Losonsky G A , Mackow E R , Greenberg H B
Published: January 2020

The humoral immune response to rhesus rotavirus (RRV) VP4 and its cleavage products VP5* and VP8* was determined in paired serum samples from 44 infants vaccinated with RRV or human rotavirus-RRV reassortants and 5 placebo recipients. Our aim was to try to measure the response to those regions of VP4 most closely related to protection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the immunoglobulin G immune response to baculovirus-expressed full-length RRV VP4, full-length VP8*, and the amino-terminal polypeptide of VP5* called VP5*(1) (amino acids 248 to 474). The two antigenic regions of VP4 selected for study, VP5*(1) and VP8*, have previously been shown to contain most of the cross-reactive and strain-specific neutralization epitopes, respectively, while the remaining carboxy-terminal half of VP5* (amino acids 475 to 776) has not been clearly associated with neutralization. All three recombinant proteins were antigenically conserved, since they reacted with a library of neutralizing monoclonal antibodies directed at VP4. There was a high percentage of seroresponders to VP4 (61%) or to VP8* (52%), but fewer infants seroresponded to VP5*(1) (11%). In addition, infants responding to VP5*(1) had considerably lower titers than to VP4 or VP8*. Immune response to VP4 correlated strongly with the responses detected by the plaque reduction neutralization assay but did not correlate with the responses detected by the ELISA to whole RRV. These data imply that the VP5*(1) region is less immunogenic than the VP8* region of VP4 in infants immunized with RRV or RRV reassortants. The low immunogenicity of VP5* might adversely affect the efficacy of RRV vaccine candidates.
Development and application of a new scheme of phages for typing and differentiating Salmonella strains from different sources
Authors: Castro D , Morińigo M A , Martinez-Manzanares E , Cornax R , Balebona M C , Luque A , Borrego J J
Published: January 2020

A set of 25 phages for typing and differentiating Salmonella strains from different origins (food, water, and disease outbreaks) is described. All the strains were typeable by use of the phages, whereas by the serological method more than 5% of the strains could not be classified. By using the phage typing scheme, 75 phage types were established, and all the results were reproducible 1 and 6 months later. Some phages were serotype and serogroup specific, which may be useful in additional tests for the identification of strains of some Salmonella serotypes. In addition, the strains responsible for an outbreak possessed the same phage type, which implies the potential epidemiological use of these phages.
Method for detection of simian immunodeficiency virus neutralizing antibodies using a noncommercial antigen capture enzyme-linked immunosorbent assay
Authors: Welch M J , Thouless M E
Published: January 2020

A neutralization test (NT) using a noncommercial antigen capture enzyme-linked immunosorbent assay (ELISA) to detect simian immunodeficiency virus (SIV) growth in vitro was developed. The capture antibody was a mixture of purified macaque anti-SIV immunoglobulin G (IgG) and a monoclonal antibody to SIV p27. Captured antigens were detected by using purified macaque anti-SIV IgG conjugated to horseradish peroxidase. The NT reliably and sensitively detected differences when various amounts of SIV were used with positive and negative control macaque sera. Dilutions of sequential sera from a macaque (Macaca nemestrina) that had been experimentally infected with SIV were tested for neutralizing antibody with 300 50% tissue culture infective doses of SIV. In this macaque, neutralizing activity and anti-SIV IgG levels in serum (detected by ELISA) increased with time after SIV inoculation, and high IgG titers were required in serum before neutralization occurred in vitro. This simple NT, which detects the presence of SIV serum neutralizing antibodies at a low cost, will be useful for investigating the role of neutralizing antibodies in the SIV-infected macaque model for AIDS.
A serotype 10 human rotavirus
Authors: Beards G , Xu L , Ballard A , Desselberger U , McCrae M A
Published: January 2020

Rotaviruses with genome rearrangements isolated from a chronically infected immunodeficient child (F. Hundley, M. McIntyre, B. Clark, G. Beards, D. Wood, I. Chrystie, and U. Desselberger, J. Virol 61:3365-3372, 1987) are the first recognized human isolates of serotype 10. This was shown by both a direct enzyme-linked immunosorbent assay and virus neutralization assays using serotype specific monoclonal antibodies. The serotype was confirmed by sequence analysis of the gene encoding VP7, which revealed a 96% amino acid homology to the bovine serotype 10 isolate B223.
Improved serological diagnosis of Toxoplasma gondii infection by detection of immunoglobulin A (IgA) and IgM antibodies against P30 by using the immunoblot technique
Authors: Gross U , Roos T , Appoldt D , Heesemann J
Published: January 2020

Immunoglobulin M (IgM) and IgA antibodies against the major surface protein of Toxoplasma gondii were determined in a total of 195 human sera and five human cerebrospinal fluids by using a P30 membrane extract and the immunoblot technique. By using two different T. gondii strains (RH and BK) simultaneously as antigens, we were able to demonstrate diagnostically important strain-specific human antibody responses in 4.5% of the samples tested. A comparison of the immunoblot technique with an IgM immunocapture enzyme-linked immunosorbent assay demonstrated that the IgM and IgA immunoblot seems to be of advantage in the diagnosis of acute toxoplasmosis in certain groups of patients, especially in the diagnosis of cerebral toxoplasmosis in patients with AIDS. The immunoblot technique described is easy to perform and might be useful as an additional serological assay for routine diagnosis of T. gondii infections.
Detection of borreliacidal antibodies in hamsters by using flow cytometry
Authors: Sachsenmeier K F , Schell K , Morrissey L W , Pennell D R , West R M , Callister S M , Schell R F
Published: January 2020

Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.
Characterization of isolates of Mycobacterium avium serotypes 4 and 8 from patients with AIDS by multilocus enzyme electrophoresis
Authors: Yakrus M A , Reeves M W , Hunter S B
Published: January 2020

Isolates of Mycobacterium avium serotypes 4 and 8 originating from patients with AIDS in New York City, Los Angeles, or San Francisco were further characterized by multilocus enzyme electrophoresis. Reference strains used to produce typing antisera were also examined. Thirty-one electrophoretic types (ETs) were found among 58 isolates of serotype 4, while 10 ETs were identified among 21 isolates of serotype 8. One major ET was found within each serotype, and these two ETs were closely related, separated by a genetic distance of only 0.05. Six ETs were found in more than one city. In four cases, isolates of serotypes 4 and 8 shared the same ET. Multilocus enzyme electrophoresis in combination with serotyping should be helpful in locating the specific infection sources of these commonly isolated opportunistic pathogens.
Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi
Authors: Jiang W , Luft B J , Schubach W , Dattwyler R J , Gorevic P D
Published: January 2020

Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.
Fatal Legionella maceachernii pneumonia in Canada
Authors: Thomas E , Gupta N K , van der Westhuizen N G , Chan E , Bernard K
Published: January 2020

A case of pneumonia and acute tubular necrosis was caused by an initially unknown species of Legionella. The organism was later identified as Legionella maceachernii by a combination of cultural, biochemical, and serological methods along with a gas-liquid chromatographic profile.
Graft-related endocarditis caused by Neosartorya fischeri var. spinosa
Authors: Summerbell R C , de Repentigny L , Chartrand C , St Germain G
Published: January 2020

The first case of endocarditis caused by Neosartorya fischeri var. spinosa is reported. The patient was a child who received a calf pericardium graft after removal of a previously inserted Dacron graft associated with deterioration of adjacent tissue. Copious vegetations removed from the heart were found to be composed of septate hyaline fungal filaments. The fungus was recognized in culture by its bivalved, winged, spiny ascospores, its Aspergillus fischerianus anamorph, and its thermotolerance.
Evaluation of microbiological processing of urine specimens: comparison of overnight versus two-day incubation
Authors: Murray P , Traynor P , Hopson D
Published: January 2020

In this study the value of urine culture incubation for 1 versus 2 days was determined. The results of the study indicate that 1 day of incubation is reliable if (i) all specimens are incubated for a full 24 h, (ii) all specimens with less than 10(4) uropathogens per ml are incubated for an additional day to ensure accurate quantitation, and (iii) specimens from patients with suspected funguria are incubated for a minimum of 2 days.
An adventitious viral contaminant in commercially supplied A549 cells: identification of infectious bovine rhinotracheitis virus and its impact on diagnosis of infection in clinical specimens
Authors: Fong C K , Landry M L
Published: January 2020

The isolation and identification of an adventitious viral agent, infectious bovine rhinotracheitis virus, in one lot of A549 cells from a commercial supplier is described in this report. The presence of infectious bovine rhinotracheitis virus in A549 cells was unexpected and has caused problems in the diagnosis of infections in clinical specimens in three laboratories.
Comparison of a modified adherence assay with existing assay methods for identification of enteroaggregative Escherichia coli
Authors: Haider K , Faruque S M , Albert M J , Nahar S , Neogi P K , Hossain A
Published: January 2020

Localized, diffuse, and aggregative adherence patterns of Escherichia coli identified with specific DNA probes were compared in cell culture adherence assays by using the Center for Vaccine Development, Baltimore, method, the University of Texas Medical School, Houston (UTH), method, and a modified UTH method. Increasing postwash incubation time from 2 to 4 h in the modified UTH method allowed identification of enteroaggregative E. coli, which was otherwise not identified by the original UTH method.
Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction
Authors: Todd D , Mawhinney K A , McNulty M S
Published: January 2020

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.
Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification
Authors: Herrmann J L , Bellenger E , Perolat P , Baranton G , Saint Girons I
Published: January 2020

Fingerprints for 72 reference serovar strains of pathogenic Leptospira spp. were obtained by pulsed-field gel electrophoresis (PFGE) following NotI restriction digests of the chromosome. These strains included the serovar reference strains of serogroups Australis, Ballum, Bataviae, Grippotyphosa, Panama, Pomona, and Pyrogenes. Sixty-four serovars could be identified by a unique NotI restriction profile. The remaining serovars were differentiated by chromosomal digestion with SgrAI. These included four serovars from serogroup Australis, two serovars from serogroup Ballum, and two serovars from serogroup Bataviae. Thirteen of 18 recent clinical isolates identified by microagglutination test and cross-adsorption procedure were correctly typed by PFGE. The results indicate that PFGE, which is considerably more rapid than serology, should be useful for identification and epidemiological studies.
Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction
Authors: Menzo S , Bagnarelli P , Giacca M , Manzin A , Varaldo P E , Clementi M
Published: January 2020

A competitive polymerase chain reaction (PCR)-based assay for the quantitative detection of human immunodeficiency virus type 1 (HIV-1) viremia was developed and optimized. This method consists of the reverse transcription and subsequent amplification in the same tube of two similar RNA templates, the wild-type template to be quantified and a known amount of the internally deleted synthetic template, both with identical primer recognition sites. The same strategy also proved to be useful in the quantitative assay of HIV-1-specific cellular transcripts and proviral DNA sequences from peripheral blood mononuclear cells by using competitor DNA. The method might be of interest in the study of the precise level of HIV-1 activity during the different clinical phases of the infection and in the simple, fast, and methodologically correct molecular investigation of patients treated with specific antiviral compounds.