bluuebunny/crossref_metadata_2025 huggingface dataset:
Confirmation of positive results for chlamydial antigen by the Chlamydiazyme assay: value of repeated testing and a blocking antibody assay
Authors: Olsen M A , Sambol A R
Published: January 2020

We studied the specificity of the Abbott Chlamydiazyme test for detection of Chlamydia trachomatis antigen by means of a specific blocking antibody test. A total of 457 previously positive specimens were tested; 22 did not block in the blocking antibody test, 39 did not repeat as positive, and 396 were confirmed as positive. The distribution of A492 values obtained with specimens which did not repeat as positive was nonrandom and was concentrated between the cutoff values and 0.400. The positive predictive value of the Chlamydiazyme assay after initial testing was 86.7% (396 of 457), but the positive predictive value increased to 94.7% (396 of 418) if specimens which were not repeatedly positive were considered negative. We recommend routinely repeating the Chlamydiazyme assay for all specimens which give A492 values between the cutoff and 0.400 to eliminate many false-positive results. Use of the blocking antibody reagent can then be reserved for confirming only specimens which are repeatedly positive.
Recovery of Borrelia burgdorferi by filtration
Authors: Jobe D A , Callister S M , Schell R F
Published: January 2020

Bacterial contamination frequently interferes with successful recovery of the Lyme spirochete from cultures of tissue from Borrelia burgdorferi-infected humans, rodents, or ticks. We used 0.20- and 0.45-microns-pore-size syringe-tip filters to recover spirochetes from cultures contaminated with other bacteria. Low concentrations (1 to 10/ml) of B. burgdorferi organisms could be recovered from cultures seeded with 1 x 10(8) to 4 x 10(8) Staphylococcus aureus, Streptococcus faecium, Escherichia coli, or Bacillus subtilis organisms per ml. We also used this technique to recover B. burgdorferi from contaminated environmental and clinical cultures of B. burgdorferi. We conclude the filtration is an efficient method for recovering Lyme spirochetes from contaminated samples and increasing the number of successful isolations of B. burgdorferi.
Comparative humoral responses to human immunodeficiency virus type 1-p24gag linear B-cell epitopes among individuals showing atypical western immunoblotting reactions and implications for diagnosis
Authors: Lallement J C , Vendrell J P , Corbeau P , Ducos J , Segondy M , Escande A , Robert-Hebmann V , Jean F , Reynes J , Serre A
Published: January 2020

Serum specimens from 25 individuals with an isolated human immunodeficiency virus type 1 (HIV-1) core antigen reactivity in a Western immunoblot test were examined for their reactivities with HIV-1 virions, control cellular antigens, HIV-1-Bru p24gag recombinant protein (p24gag), and a panel of 22 p24gag-derived peptides. The results were as follows: (i) serum specimens from eight HIV-1-uninfected subjects did bind to virions but failed to bind to p24gag; (ii) sera from 13 HIV-1-uninfected subjects and from one HIV-2-infected patient reacted with HIV-1 virions and p24gag but failed to bind to any of the peptides expressing major p24gag epitopes, and (iii) 3 serum specimens obtained from one neonate carrying anti-HIV-1 maternal antibody and from two HIV-1-infected subjects who had seroconverted during the study reacted with HIV-1 virions, p24gag, and one or more peptides containing the major p24gag epitopes. Our data suggest that the combination of p24gag and appropriate peptides could be useful for resolution when atypical Western immunoblot results are encountered.
Synergy characterization for Enterococcus faecalis strains displaying moderately high-level gentamicin and streptomycin resistance
Authors: Bantar C E , Micucci M , Fernandez Canigia L , Smayevsky J , Bianchini H M
Published: January 2020

Synergy of 14 Enterococcus faecalis strains displaying moderately high-level aminoglycoside resistance (MICs, 500 and 256 to 1,000 micrograms/ml for gentamicin and streptomycin, respectively) was characterized by time-kill studies. All strains proved resistant to penicillin plus the respective aminoglycoside. Strains with moderately high-level aminoglycoside resistance should be considered to exhibit high-level resistance in severe infections.
Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates
Authors: Woods C R , Versalovic J , Koeuth T , Lupski J R
Published: January 2020

Repetitive element sequence-based polymerase chain reaction (rep-PCR) enables the generation of DNA fingerprints which discriminate bacterial species and strains. We describe the application of whole-cell methods which allow specimens from broth cultures or colonies from agar plates to be utilized directly in the PCR mixture. The rep-PCR-generated DNA fingerprints obtained with whole-cell samples match results obtained with genomic DNA templates. Examples with different gram-negative bacteria (e.g., Citrobacter diversus, Escherichia coli, and Pseudomonas aeruginosa) and gram-positive bacteria (e.g., Staphylococcus aureus and Streptococcus pneumoniae) are demonstrated. Rapid specimen preparation methods enable rep-PCR-based fingerprinting to be completed in several hours and, therefore, allows the timely analysis of epidemiological relationships.
Use of immunoelectron microscopy to demonstrate Francisella tularensis
Authors: Geisbert T W , Jahrling P B , Ezzell J W
Published: January 2020

Three immunoelectron microscopy (IEM) methods were employed to show laboratory-cultivated Francisella tularensis. By the IEM assays, F. tularensis was distinguished from four antigenically distinct gram-negative bacteria. IEM should be a valuable tool for confirming presumptive isolates of F. tularensis and may potentially be useful for demonstrating other medically important bacteria.
Human cytomegalovirus (HCMV) late-mRNA detection in peripheral blood of AIDS patients: diagnostic value for HCMV disease compared with those of viral culture and HCMV DNA detection
Authors: Gozlan J , Salord J M , Chouaïd C , Duvivier C , Picard O , Meyohas M C , Petit J C
Published: January 2020

A reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect a human cytomegalovirus (HCMV) late mRNA in peripheral blood leukocytes (PBL) of 102 human immunodeficiency virus-infected individuals. The clinical value of this new technique for the diagnosis of acute HCMV disease was evaluated in comparison with viral culture and direct amplification of viral DNA (PCR). The sensitivity of the RT-PCR was slightly lower than that of the two other methods, but its specificity was 94%, compared to 55 and 32% for culture and PCR, respectively. Transcription of this late mRNA is linked to viral replication, and its detection in PBL confirms that these cells can support a complete viral cycle. The relationship between complete replicative cycles and HCMV disease makes RT-PCR a useful clinical tool.
Concordant clonal delineation of methicillin-resistant Staphylococcus aureus by macrorestriction analysis and polymerase chain reaction genome fingerprinting
Authors: Struelens M J , Bax R , Deplano A , Quint W G , Van Belkum A
Published: January 2020

Pulsed-field gel electrophoresis of DNA macrorestriction fragments (macrorestriction analysis) allows epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) by indexing variations in the global chromosome architecture. Polymerase chain reaction (PCR)-mediated genome fingerprinting can also discriminate MRSA strains by detecting locally variable DNA motifs. To assess the correlation between these methods, 48 epidemic MRSA strains collected from 20 hospitals over a 10-year period were tested in a blind comparison by (i) macrorestriction analysis with SstII or SmaI endonuclease and (ii) PCR fingerprinting with four primer sets aimed at the mecA gene, enterobacterial repetitive intergenic consensus sequences, and arbitrary sequences. Isolates were discriminated into 22 macrorestriction patterns and 15 PCR fingerprints. MRSA strains belonging to 12 distinct clones by macrorestriction analysis showed 11 distinct PCR genotypes distinguished by multiple band differences. In contrast, 34 of 37 MRSA strains found to be clonally related by macrorestriction analysis clustered in two highly related PCR genotypes that differed by a single DNA fragment (P < 0.0001). These data demonstrate concordant clonal delineation of epidemic MRSA by macrorestriction analysis and PCR fingerprinting and thereby indicate that the rapid PCR assay may be an efficient epidemiologic typing system.
Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains
Authors: Jallat C , Livrelli V , Darfeuille-Michaud A , Rich C , Joly B
Published: January 2020

Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E. coli involved in diarrheal diseases. A total of 1.5% of them belonged to the enterotoxigenic E. coli pathotype, but none belonged to the enteroinvasive E. coli, enterohemorrhagic E. coli, or enteropathogenic E. coli pathotypes. Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe. Most of the strains involved in diarrhea belonged to the diffusely adhering E. coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells. Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells. The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E. coli strains should be considered pathogens. Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe. Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe. In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane. This cytotoxic effect was correlated with the synthesis of a hemolysin. The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.
Evaluation of genetic divergence of borrelial isolates from Lyme disease patients in Hokkaido, Japan, by rRNA gene probes
Authors: Fukunaga M , Sohnaka M , Nakao M , Miyamoto K
Published: January 2020

Eight spirochetal isolates (JEM1 to JEM8) were obtained from cutaneous lesions of patients with Lyme disease in Hokkaido, Japan, and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reactivities with monoclonal and polyclonal antibodies, and Southern blot hybridization. The protein profiles of these borrelial isolates were variable and differed markedly from that of Borrelia burgdorferi B31. The 41-kDa flagellin protein was present in all isolates, but the outer surface protein A that reacted with monoclonal antibody H5332 was absent from four clinical isolates (JEM1, JEM5, JEM7, and JEM8). Genomic hybridization with rRNA gene probes demonstrated the genetic divergences among those isolates. These findings indicate that the borrelial isolates from patients in Japan are quite characteristically unique.
Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory
Authors: Clarridge J E , Shawar R M , Shinnick T M , Plikaytis B B
Published: January 2020

We investigated the use of DNA amplification by the polymerase chain reaction reaction (PCR) for detection of Mycobacterium tuberculosis from clinical specimens. Two-thirds of each sample was processed for smear and culture by standard methods, and one-third was submitted for DNA extraction, amplification of a 317-bp segment within the insertion element IS6110, and detection by agarose gel electrophoresis, hybridization, or both. DNA was prepared from over 5,000 samples, with 623 samples being culture positive for acid-fast bacilli. Of 218 specimens that were identified as M. tuberculosis, 181 (85%) were positive by PCR. In the M. tuberculosis culture-positive group, PCR was positive for 136 of 145 (94%) and 45 of 73 (62%) of the fluorochrome smear-positive and -negative specimens, respectively. Of 948 specimens that were either culture positive for mycobacteria other than M. tuberculosis or culture negative, 937 specimens were negative by PCR and 11 (1%) specimens initially appeared to be false positive for M. tuberculosis. The reason for discrepant results varied; some errors were traced to the presence of an inhibitor in the specimen (7.3% in unselected specimens), nucleic acid contamination, low numbers of organisms in the specimen antituberculosis therapy, and possible low-level nonspecific hybridization. In comparison with culture, the sensitivity, specificity, and positive predictive value were 83.5, 99.0, and 94.2%, respectively, for PCR. When PCR was corrected for DNA contamination, the presence of inhibitor, and culture-negative disease, the values became 86.1, 99.7, and 98.4%, respectively. If the results for multiple specimens submitted from the same patient are considered, no patient who had three of more sputum specimens tested would have been misdiagnosed.
Rapid diagnosis of scrub typhus by a passive hemagglutination assay using recombinant 56-kilodalton polypeptides
Authors: Kim I S , Seong S Y , Woo S G , Choi M S , Kang J S , Chang W H
Published: January 2020

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes of Rickettsia tsutsugamushi, Gilliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were evaluated with 89 serum specimens from health blood donors, 94 serum specimens from scrub typhus patients, and 31 serum specimens from patients with other febrile diseases by a passive hemagglutination assay (PHA). Among the scrub typhus patients diagnosed by indirect immunofluorescent-antibody testing, the antibodies to R. tsutsugamushi were detected in 93 patients (99%). One serum specimen from a healthy person showed a false-positive reaction by this method. The recombinant PHA showed no cross-reactions with sera obtained from other febrile patients with diseases such as murine typhus, hemorrhagic fever with renal syndrome, and leptospirosis. In conclusion, this recombinant PHA could be substituted for the conventional indirect immunofluorescent-antibody test and the immunoperoxidase test.
Twelve aberrant strains of Staphylococcus aureus subsp. aureus from clinical specimens
Authors: Fontana C , Cellini L , Dainelli B
Published: January 2020

A new biovar of Staphylococcus aureus subsp. aureus was isolated from human clinical specimens and described on the basis of studies of 12 isolates that were compared with 11 standard reference strains. Both DNA hybridization experiments and numerical taxonomy analysis demonstrated that these strains were strictly related to S. aureus subsp. aureus; however, they were significantly different from the latter. The atypical strains belonging to the new biovar can be distinguished from typical S. aureus subsp. aureus strains by their alpha-chymotrypsin, alpha-glucosidase, beta-N-acetylglucosaminidase, lipase (C-14), and leucine arylamidase enzymatic activities and novobiocin resistance. Thus, the combination of alpha-glucosidase and beta-N-acetyl-glucosaminidase is more useful for distinguishing these S. aureus strains from the other, typical ones.
Controlled clinical comparison of two lysis-based blood culture systems, isolator and Septi-Chek Release, for detection of bloodstream infections
Authors: Kirkley B A , Easley K A , Basille B A , Washington J A
Published: January 2020

A controlled clinical comparison was made of the Isolator (Wampole Laboratories, Cranbury, N.J.) and the Septi-Chek Release bottle (Roche Diagnostics, Nutley, N.J.). From 6,345 blood culture sets fulfilling minimum criteria for volume of blood cultured, 840 strains were isolated, of which only 691 (82%) were considered to be representative of bloodstream infection according to Centers for Disease Control definitions. Statistically significant differences were found between the systems for the following organisms, which were all detected more frequently in the Isolator system: Staphylococcus aureus (P = 0.0001), Alcaligenes xylosoxidans (P = 0.008), Klebsiella pneumoniae (P = 0.05), Salmonella spp. (P = 0.03), and Candida albicans (P = 0.02). The Septi-Chek Release system required a longer period of time than the Isolator system for detection of the following organisms:S. aureus (P = 0.0001), Enterococcus spp. (P = 0.0001), Enterobacter cloacae (P = 0.03), Escherichia coli (P = 0.0001), Klebsiella oxytoca (P = 0.03), K. pneumoniae (P = 0.02), Pseudomonas aeruginosa (P = 0.002), and C. albicans (P = 0.005). There were 430 episodes of bloodstream infections identified in the study; of these episodes, only those due to S. aureus were detected significantly more frequently (P = 0.0001) by the Isolator system than by the Septi-Chek Release system. However, episodes of bloodstream infections due to S. aureus, Staphylococcus epidermidis, Enterococcus spp., and E. coli were detected significantly faster by the Isolator system.
Characterization of a chromosomal gene and the antigen it expresses from the Lyme disease agent, Borrelia burgdorferi
Authors: LeFebvre R B , Probert W S , Perng G C
Published: January 2020

The sequence and characterization of a chromosomal gene from the Lyme disease agent Borrelia burgdorferi and the antigen it encodes are described. The gene was cloned and expressed in transformed Escherichia coli cells. The gene is composed of 597 bases and expresses a predicted protein of 199 amino acids. Antibodies specific for the recombinant antigen reacted with a single B. burgdorferi protein with a molecular mass of approximately 22 kDa. The protein was not susceptible to proteinase digestion but was extracted by n-butanol phase partitioning, suggesting a periplasmic location of the antigen. Sera from humans and canines seropositive for B. burgdorferi reacted with the recombinant antigen. The antigen characterized in this report appears to be immunologically significant in naturally infected hosts.
A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates
Authors: Li C , Ferguson D A , Ha T , Chi D S , Thomas E
Published: January 2020

HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies.
Detection of immunoglobulin G antibodies to Helicobacter pylori in urine by an enzyme immunoassay method
Authors: Alemohammad M M , Foley T J , Cohen H
Published: January 2020

Urine and serum samples from 306 patients undergoing upper endoscopy were evaluated prospectively to determine the presence of immunoglobulin G (IgG) antibodies to Helicobacter pylori by an enzyme immunoassay method. Forty-nine selected urine specimens were also tested by Western blotting (immunoblotting). When compared with bioptic methods (culture, stain, urease testing), the sensitivity and specificity of the assay for urine IgG to H. pylori were 95.9 and 90%, respectively. Results of testing of serum and urine for IgG to H. pylori were concordant for 95% of samples. Western blot analysis revealed a highly variable antibody response to H. pylori antigens among patients. Detection of IgG antibody to H. pylori in urine is simple and reflects the presence or absence of H. pylori infection.
Typing of Legionella pneumophila strains by polymerase chain reaction-mediated DNA fingerprinting
Authors: van Belkum A , Struelens M , Quint W
Published: January 2020

Well-defined Legionella pneumophila strains were analyzed by amplification of variable genomic regions with arbitrary and repeat sequence primers. Clinical and environmental outbreak-related isolates showed closely related amplicon patterns. Eleven strains of unrelated origins displayed 10 distinct patterns. Fingerprinting of L. pneumophila by polymerase chain reaction appeared to have the potential of being as epidemiologically useful as other genotypic methods.
Isolation of Escherichia fergusonii from four different sites in a patient with pancreatic carcinoma and cholangiosepsis
Authors: Funke G , Hany A , Altwegg M
Published: January 2020

Escherichia fergusonii was isolated from a 69-year-old male with pancreatic carcinoma and cholangiosepsis from gallbladder fluid, three blood cultures, feces, and a superficial wound of the abdomen. Biochemical reactions, antimicrobial susceptibility patterns, susceptibility to polyvalent phage 0-1, and rRNA gene restriction analysis suggested that the four strains were of clonal origin. Our data indicate that E. fergusonii possesses a pathogenic potential in humans.
Prevalence of antibodies to spotted fever group rickettsiae along the eastern coast of the Adriatic sea
Authors: Radulovic S , Walker D H , Weiss K , Dzelalija B , Morovic M
Published: January 2020

A seroepidemiological survey in coastal Croatia detected antibodies reactive with Rickettsia conorii in 4.2% of sera by immunofluorescence assay and in 5.0% of sera by enzyme immunoassay. Western immunoblotting demonstrated antibodies to the 120-kDa surface protein in all 20 positive serum samples examined and to rickettsial lipopolysaccharide in 3 of these serum samples. Humans in this area are clearly being exposed to spotted fever rickettsiae.
Evaluation of molecular typing for epidemiological study of Chlamydia trachomatis genital infections
Authors: Rodriguez P , de Barbeyrac B , Persson K , Dutilh B , Bebear C
Published: January 2020

Molecular typing and serotyping were compared for 150 Chlamydia trachomatis strains isolated from genital sources, belonging to 10 different serovars. Because of the general agreement of the two methods, molecular omp1 genotyping was applied to the epidemiological study of C. trachomatis isolates from genital infections in Bordeaux (France), during a 29-month period. The most prevalent omp1 genotypes were E (51.7%), F (17.3%), D (8.8%), and G (8.4%). Restriction enzyme analysis allowed identification of a serovar D variant (Dv), whereas serovar E strains were homogeneous.
Gene probes for identification of the botulinal neurotoxin gene and specific identification of neurotoxin types B, E, and F
Authors: Campbell K D , Collins M D , East A K
Published: January 2020

A polymerase chain reaction method was developed for the specific detection of the botulinum neurotoxin (BoNT) gene of Clostridium botulinum. Degenerate oligonucleotide primers, designed from the nucleotide sequence of the heavy chain of the BoNT gene, amplified a specific fragment of approximately 1.1 kb from strains of C. botulinum toxin types A, B, E, F, and G and neurotoxin-producing strains of Clostridium barati and Clostridium butyricum, but no fragment was obtained from nontoxigenic strains. The fragments amplified from several strains of C. botulinum types B, E, and F were cloned in Escherichia coli and their nucleotide sequences were determined. Sequences within this region were used to design oligonucleotide probes specific for BoNT type B (BoNT/B), BoNT/E, and BoNT/F genes. An additional probe was designed for the detection of the BoNT/F gene of C. barati, which differed in sequence from BoNT/F genes of both proteolytic and nonproteolytic strains of C. botulinum.
Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus
Authors: Kang S Y , Benfield D A , Gorziglia M , Saif L J
Published: January 2020

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.
Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis
Authors: Verschoor E J , van Vliet A L , Egberink H F , Hesselink W , Horzinek M C , de Ronde A
Published: January 2020

The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleavage occurred in Sf9 cells expressing the envelope precursor, but further processing was not observed. Crude lysates of insect cells infected with the wild-type baculovirus or with the recombinant viruses were used to develop an enzyme-linked immunosorbent assay for the detection of feline immunodeficiency virus-specific antibodies in cat sera. The assay showed a higher sensitivity and specificity than immunofluorescence and Western blotting (immunoblotting).
Influence of zinc on Pseudomonas aeruginosa susceptibilities to imipenem
Authors: Cooper G L , Louie A , Baltch A L , Chu R C , Smith R P , Ritz W J , Michelsen P
Published: January 2020

Serial dilution susceptibility testing of imipenem against 59 clinical isolates of Pseudomonas aeruginosa, conducted simultaneously on single lots of Difco and BBL Mueller-Hinton agar (MHA), resulted in MICs for 90% of strains tested of 8 and 16 micrograms/ml, respectively. MICs for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas spp. were also higher on BBL MHA. Quantification of the cation content of the two MHAs by atomic absorption spectroscopy demonstrated that the zinc concentration in BBL MHA was 15 times greater than that measured in Difco MHA (2.61 and 0.17 micrograms/ml, respectively). Concentrations of calcium, magnesium, iron, manganese, and copper in the two agars were similar. Addition of zinc to Difco MHA resulted in increases in MICs of imipenem for P. aeruginosa but not in the MICs of ceftazidime or cefpirome for P. aeruginosa (P < 0.01). A lesser zinc effect was seen on the activity of imipenem against E. coli, K. pneumoniae, and Pseudomonas spp. The activities of ceftazidime and cefpirome were similar on both MHAs when tested against all gram-negative organisms in this study. Thus, the effect of zinc in MHA was clearly demonstrated by a significant increase in the MICs of imipenem for P. aeruginosa, and, to a lesser extent, for other gram-negative bacilli.
Evaluation of sputum smears concentrated by cytocentrifugation for detection of acid-fast bacilli
Authors: Saceanu C A , Pfeiffer N C , McLean T
Published: January 2020

Early identification and isolation of tuberculosis patients is of utmost importance to minimize the risk of further epidemic spread of the disease. The traditional concentrated acid-fast smears are not very reliable tools for the presumptive diagnosis of tuberculosis. Acid-fast bacillus (AFB) smears from 120 patients specimens and 80 simulated AFB samples were processed according to standard laboratory procedures and by cytocentrifugation (Cyto-Tek, Ames Division, Miles Laboratories, Inc., Elkhart, Ind.). Prior to dispensing of samples into the Cyto-Tek chambers, specimens were liquefied and decontaminated by mixture with an equal volume of 5% sodium hypochlorite (household bleach). Culture and smear results were correlated. Of 120 patient specimens, 43 were culture and smear negative by both methods. Ten specimens were overgrown with mold and bacteria, but 2 of them had positive AFB smears by cytocentrifugation only. There were 67 positive AFB cultures, with 67 positive cytocentrifuge smears and 34 positive smears by the conventional technique. Of the 80 simulated positive AFB samples, all grew mycobacteria on culture. Smears from the 10(5)- to 10(3)-CFU/ml specimens were positive by both methods. The simulated samples with 10(2) CFU/ml yielded smears positive only by cytocentrifugation. The Cyto-Tek AFB smears had a greater correlation with positive culture than did the smears from concentrated specimens. The sensitivity, efficiency, and rapidly of the Cyto-Tek AFB smear technique resulted in increased detection of mycobacteria in clinical specimens. The simplicity and safety of this method will enable qualified mycobacteriology technologists to rapidly and accurately perform sputum smears for AFB at clinics, emergency rooms, and field sites, as well as in the traditional laboratory setting.
Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA
Authors: Jonas V , Alden M J , Curry J I , Kamisango K , Knott C A , Lankford R , Wolfe J M , Moore D F
Published: January 2020

Seven hundred fifty-eight processed sputum sediments received for the diagnosis of tuberculosis or other mycobacterial infections were tested by utilizing a rRNA target amplification assay and traditional culture techniques. The results from the rRNA target amplification assay (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test), available in 5 h, were compared with the results from standard culture techniques held for 6 weeks. A total of 119 specimens (16%) were culture positive for Mycobacterium tuberculosis. Overall sensitivity, specificity, positive predictive value, and negative predictive value were 82, 99, 97, and 96%, respectively, for the Gen-Probe assay; 88, 100, 100, and 97%, respectively, for culture; and 53, 99.8, 99.6, and 91%, respectively, for fluorochrome stain. The Gen-Probe assay employs the isothermal enzymatic amplification of M. tuberculosis complex rRNA followed by detection of the amplicon with an acridinium ester-labeled DNA probe. This assay has the potential of reducing the time for diagnosis of tuberculosis to 1 day.
Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis
Authors: Avila H A , Pereira J B , Thiemann O , De Paiva E , DeGrave W , Morel C M , Simpson L
Published: January 2020

A panel of 114 blood samples from chronic chagasic patients and nonchagasic patients was screened for Trypanosoma cruzi by xenodiagnostic, serologic, and polymerase chain reaction (PCR) amplification tests. Blood samples were preserved in a guanidine-EDTA buffer, and total blood DNA was isolated after chemical nuclease cleavage with 1,10-phenanthroline-copper ion and used as a template for PCR amplification of the conserved and variable regions of T. cruzi minicircle molecules. The PCR products were screened by Southern blot hybridization with a digoxigenin-labeled oligonucleotide probe specific for the conserved region of the minicircle. The method showed a sensitivity of 100% compared with the serologic test. In addition, all of the serology-positive, xenodiagnosis-negative samples were positive by PCR. This demonstrates that PCR amplification of T. cruzi kinetoplast minicircle DNA could replace xenodiagnosis for evaluation of parasitemia in chronic chagasic patients and could serve as a complement for serologic testing in the screening of blood bank donors.
Intrathecal immune response and virus-specific immunoglobulin M antibodies in laboratory diagnosis of acute poliomyelitis
Authors: Roivainen M , Agboatwalla M , Stenvik M , Rysä T , Akram D S , Hovi T
Published: January 2020

The intrathecal immune response in 114 patients with clinically diagnosed acute poliomyelitis was studied by measuring poliovirus-specific immunoglobulin M (IgM) antibodies in cerebrospinal fluid (CSF) by a mu-capture immunoassay and by assessing the ratio between levels of poliovirus-neutralizing antibodies in serum and CSF. Fecal specimens were used for attempts to isolate the causative agents. Eighty-five percent of CSF specimens collected during the first 15 days of disease contained virus-specific IgM antibodies. Forty-five of 48 tested children (94%) also showed virus-specific IgM responses in their sera. Later on, the antibody levels decreased, and positive results after 30 days of onset of paralytic symptoms were rare. If the presence of poliovirus-specific IgM antibodies in the CSF was considered diagnostic, more cases were confirmed by this test than by virus isolation. A relative increase in poliovirus-neutralizing antibodies in the CSF was observed in about one-third of the cases; in all but three cases the increase was observed together with the presence of virus-specific IgM antibodies. A systemic virus-specific response can be seen and poliovirus can be isolated from a subclinically infected individual suffering from a concomitant poliomyelitis-like disease, while positive results by the two methods demonstrating an intrathecal immune response are likely to indicate a true causal relationship between infection and disease. Demonstration of poliovirus-specific IgM antibodies in the CSF thus appears to be a sensitive and specific method for laboratory confirmation of clinically diagnosed poliomyelitis.
Large-scale DNA fingerprinting of Mycobacterium tuberculosis strains as a tool for epidemiological studies of tuberculosis
Authors: Chevrel-Dellagi D , Abderrahman A , Haltiti R , Koubaji H , Gicquel B , Dellagi K
Published: January 2020

We conducted a large-scale DNA fingerprinting analysis of Mycobacterium tuberculosis strains in a country in which tuberculosis is endemic (Tunisia) in order to evaluate the importance of microepidemics in the maintenance of the disease within the population. The genetic polymorphisms of 201 strains of M. tuberculosis isolated from 196 unrelated patients living in four districts of northern Tunisia during a 3-year period were studied by restriction fragment length polymorphism (RFLP) analysis by using the insertion sequence IS6110 as a probe. Seventy-three strains isolated from 68 patients living in the districts of Tunis, Nabeul, and Jendouba generated 67 different RFLPs, indicating a high degree of polymorphism of the M. tuberculosis strains within these areas. In contrast, the 128 strains isolated from individuals in the district of Menzel Bourguiba appeared much less heterogeneous since they often generated identical or very similar fingerprints. Seventeen of 29 cases (58%) of active tuberculosis in the city of Menzel Bourguiba could be traced to as few as four M. tuberculosis strains. These results indicate the persistence of underestimated microepidemics in this region. The RFLP typing of a large number of randomly collected strains provides a general picture of the strains involved in tuberculosis. The systematic study of limited areas where tuberculosis is endemic can provide evidence for the existence of persisting epidemics. This stresses the different problems which remain to be solved in order to improve the control of tuberculosis.
Neisseria weaveri sp. nov., formerly CDC group M-5, a gram-negative bacterium associated with dog bite wounds
Authors: Andersen B M , Steigerwalt A G , O'Connor S P , Hollis D G , Weyant R S , Weaver R E , Brenner D J
Published: January 2020

CDC group M-5 is a rod-shaped, gram-negative, nonmotile bacterium associated with dog bite wounds. DNA-DNA relatedness and biochemical and growth characteristics were studied for 54 strains from the collection at the Centers for Disease Control and Prevention. One typical M-5 strain, 8142, was further studied by 16S rRNA sequencing. DNA from 40 of 53 strains showed 82 to 100% relatedness (hydroxyapatite method) to labeled DNA from strain 8142. The guanine-plus-cytosine (G + C) content in 8 of the 41 highly related M-5 strains was 50.5 to 52 mol%. These 41 strains were oxidase and catalase positive, nonfermentative, nitrite positive, nitrate negative, weakly phenylalanine deaminase positive, aerobic, and alpha-hemolytic (sheep blood). DNA from the 13 remaining strains showed only 7 to 46% DNA relatedness to strain 8142. These 13 non-M-5 strains differed from the M-5 strains in G + C content, growth characteristics, and biochemical profiles. DNA from M-5 strain 8142 was most closely related to DNA from groups EF-4b (47%) and EF-4a (45%). 16S rRNA sequence analysis placed M-5 strain 8142 in the Neisseriaceae cluster of the beta-3 subgroup of the class Proteobacteria. It was most homologous (98.4 to 98.8%) to Neisseria animalis, Neisseria flavescens, Neisseria canis, and Neisseria elongata. All data are consistent with M-5 being a new species of Neisseria, for which we propose the name Neisseria weaveri.
Typing of Aspergillus species and Aspergillus fumigatus isolates by interrepeat polymerase chain reaction
Authors: van Belkum A , Quint W G , de Pauw B E , Melchers W J , Meis J F
Published: January 2020

Polymerase chain reaction amplification of repetitive DNA motifs allows discrimination of Aspergillus species. In a preliminary survey, the DNA fingerprints appear to be identical when isolates of Aspergillus fumigatus are compared. When a primer deduced from a prokaryotic repeat motif is used, Aspergillus fumigatus isolates originating from different patients or different anatomical locations can be typed individually.
Clonal relationships among classical Vibrio cholerae O1 strains isolated between 1961 and 1992 in Bangladesh
Authors: Faruque S M , Abdul Alim A R , Rahman M M , Siddique A K , Sack R B , Albert M J
Published: January 2020

In Bangladesh, the replacement of classical Vibrio cholerae by the E1 Tor biotype in 1968 and the reappearance of the classical biotype and its coexistence with the E1 Tor biotype after 1982 were never adequately explained. We have analyzed 23 classical V. cholerae isolates collected between 1961 and 1968, 14 classical isolates collected between 1982 and 1992 from the capital city, Dhaka, and 6 classical V. cholerae isolates collected from two southern districts of Bangladesh and studied restriction endonuclease cleavage patterns of rRNA genes (ribotypes) to investigate the clonal relationships among the isolates. Southern blots of total DNA digested with restriction enzyme BamHI, BglI, EcoRI, HindIII, or PstI were probed, using a cloned Escherichia coli rRNA operon. While restriction enzymes BamHI, EcoRI, and PstI failed to differentiate the isolates on the basis of ribotyping, BglI and HindIII produced digestion patterns that allowed differentiation. Ribotyping the isolates with BglI and HindIII revealed five different clones (ribotypes IA, IB, IIA, IC, and IIC) of classical vibrios in Bangladesh. Strains belonging to ribotypes IA and IB were isolated in Dhaka before 1968, and one ribotype (IA) was again isolated between 1982 and 1992. Ribotype IIA was isolated in 1988 and 1989, when both clones (IA and IIA) of classical vibrios coexisted with the EI Tor vibrios. Isolates belonging to ribotypes IC and IIC were collected in the southern districts of Bangladesh and were clearly different from those collected in Dhaka between 1968 and 1992 by ribotyping analysis with BglI. These results support the previous assumption that classical vibrios were never completely replaced in Bangladesh and also demonstrate the existence of more than one genetically different clone of classical V. cholerae.
Cytopathic effect mimicking virus culture due to Mycobacterium tuberculosis
Authors: Gadea I , Zapardiel J , Ruiz P , Gegúndez M I , Esteban J , Soriano F
Published: January 2020

Mycobacterium tuberculosis grew, from a bronchoalveolar lavage of a patient with AIDS, on a gentamicin-supplemented cell culture monolayer, causing a focal and slowly spreading cytopathic effect resembling that of a virological isolate. The same effect was observed after inoculation of two different inocula of M. tuberculosis onto the same cell culture.
False-negative cerebrospinal fluid cryptococcal latex agglutination tests for patients with culture-positive cryptococcal meningitis
Authors: Currie B P , Freundlich L F , Soto M A , Casadevall A
Published: January 2020

Three cases of false-negative cerebrospinal fluid latex agglutination test results for patients with culture-positive cryptococcal meningitis are reported. False-negative results occurred in settings of low cryptococcal antigen concentrations in cerebrospinal fluid and were dependent on the latex agglutination test kit used. Investigation of each case revealed that prozone phenomena or interference from bound antibody or protein could not account for the false-negative results.
Mucor ramosissimus Samutsevitsch isolated from a thigh lesion
Authors: Weitzman I , Della-Latta P , Housey G , Rebatta G
Published: January 2020

Mucor ramosissimus Samutsevitsch is presented for the first time as an etiologic agent of cutaneous zygomycosis in a patient with aplastic anemia on immunosuppressive therapy. This report also represents the third case caused by this species reported in the literature. A biopsy taken from a lesion on the patient's thigh revealed broad, nonseptate, nonbranching hyphae compatible in morphology with a Zygomycete; M. ramosissimus was cultured twice from the thigh lesion. The patient was treated successfully with amphotericin B. Identifying features of M. ramosissimus include the following: numerous sporangia lacking columellae and resembling those of Mortierella spp., short, erect sporangiophores repeatedly branching sympodially; tough, persistent, and diffluent sporangial walls; numerous oidia in chains; extremely low colonies; and restricted growth at 36 degrees C. This paper describes the isolate and strives to alert the clinical microbiologist to this rarely reported pathogen.
Use of an enzyme-linked immunoassay for Clostridium difficile serogrouping
Authors: Delmée M , Depitre C , Corthier G , Ahoyo A , Avesani V
Published: January 2020

An enzyme-linked immunoassay (ELISA) with 11 Clostridium difficile serogroup-specific antisera was applied for serogrouping of C. difficile colonies from 314 consecutive positive fecal samples. Two hundred forty-nine strains (79%) were correctly serogrouped, 57 (18%) belonged to serogroups other than the 11 which were evaluated and gave a negative reaction with all antisera, and 8 isolates (2.5%) did not react with their corresponding antisera. ELISA is a rapid and reliable method for serogrouping C. difficile and should allow for the automation of this procedure.
Severe human infections caused by Vibrio metschnikovii
Authors: Hansen W , Freney J , Benyagoub H , Letouzey M N , Gigi J , Wauters G
Published: January 2020

Vibrio metschnikovii is largely distributed in the aquatic environment; human infections are rarely observed. A fatal case of septicemia in a patient with liver cirrhosis, renal insufficiency, and diabetes is described. A second case in a 82-year-old woman with septicemia, respiratory problems, and infected leg lesions is reported; she was successfully treated.
Reliability of cord formation in BACTEC media for presumptive identification of mycobacteria
Authors: Morris A J , Reller L B
Published: January 2020

We evaluated cord formation in BACTEC media as a criterion for presumptive identification of Mycobacterium tuberculosis complex. Auramine-rhodamine smears from 673 radiometrically positive BACTEC vials were examined. The presence of cording had a sensitivity, a specificity, and positive and negative predictive values of 22.9, 98.5, 82.9, and 80.3%, respectively. Before cord formation is used to report presumptive identification of M. tuberculosis complex, workers should determine the reliability of this finding in their own laboratories.
Incidence of Neisseria gonorrhoeae isolates negative by Syva direct fluorescent-antibody test but positive by Gen-Probe accuprobe test in a sexually transmitted disease clinic population
Authors: Beebe J L , Rau M P , Flageolle S , Calhoon B , Knapp J S
Published: January 2020

To determine the accuracy of the Syva (Palo Alto, Calif.) direct fluorescent-antibody (DFA) test in comparison with the Gen-Probe (San Diego, Calif.) Accuprobe culture confirmation test, we tested 395 isolates of Neisseria gonorrhoeae from cultures obtained from patients attending a sexually transmitted disease clinic from 1 July 1991 through 30 June 1992. All isolates were tested for DFA reactivity with a polyclonal reagent (Difco Laboratories, Detroit, Mich.) and a monoclonal reagent (Syva, Inc., direct specimen test) and for specific molecular probe reactivity by the Gen-Probe Accuprobe culture confirmation test for N. gonorrhoeae. The 395 isolates gave positive results for the Gen-Probe culture confirmation test and the Difco polyclonal direct specimen test. However, 18 (4.6%) of the isolates were negative for N. gonorrhoeae by the Syva DFA test. With the exception of six beta-lactamase-positive isolates, all isolates that were negative by Syva DFA were sensitive to penicillin, tetracycline, spectinomycin, and ceftriaxone by disk-diffusion susceptibility testing. Auxotyping and serotyping studies indicated that strains negative by Syva DFA consisted of several variants. The frequency of N. gonorrhoeae isolates showing negative results by Syva DFA in this patient population ranged from 0 to 11.5%/month. Laboratories using only the Syva DFA test for confirmation of N. gonorrhoeae may incur a significant risk of misidentification.