bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors influencing the infectivity of Chlamydia pneumoniae elementary bodies on HL cells
Authors: Theunissen J J , van Heijst B Y , Wagenvoort J H , Stolz E , Michel M F
Published: January 2020

The influence of variations in the pH, NaCl concentration, temperature, and concentrations of calcium and magnesium ions on the survival of Chlamydia pneumoniae elementary bodies (EBs) outside the host cells was investigated. The survival was determined after various incubation periods by counting the inclusion-forming units after C. pneumoniae was cultured for 72 h on monolayers of HL cells. The normal physiological conditions were restored prior to infecting the HL cells with C. pneumoniae. Declines in the infectivities of C. pneumoniae EBs were observed at pH values of lower than 5 and higher than 8 or at NaCl concentrations of less than 80 mM. The viability of C. pneumoniae EBs in SPG medium decreased as the temperature and/or incubation period increased. Incubation temperatures of up to 20 degrees C and incubation periods of up to 48 h did not affect the viability of C. pneumoniae. One hundred percent of the C. pneumoniae EBs were infective after 1 h of incubation at 35 degrees C, whereas 90, 50, and 40% survived after incubations of 8, 24, and 48 h, respectively. The viability of C. pneumoniae was unaffected within the investigated range of Ca2+ and Mg2+ ion concentrations in the medium. The presence of 10% fetal calf serum in the incubation medium had a stabilizing effect on the viability of C. pneumoniae. This effect became more pronounced as the incubation period increased.
High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms
Authors: De Briel D , Couderc F , Riegel P , Jehl F , Minck R
Published: January 2020

A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory.
Antibodies to Borrelia burgdorferi in rodents in the eastern and southern United States
Authors: Magnarelli L A , Oliver J H , Hutcheson H J , Boone J L , Anderson J F
Published: January 2020

Serologic studies were conducted to determine whether white-footed mice (Peromyscus leucopus) and cotton mice (Peromyscus gossypinus) contained serum antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Enzyme-linked immunosorbent assays detected antibodies to this spirochete in 35.7 and 27.3% of 56 P. leucopus and 535 P. gossypinus serum samples, respectively, collected in Connecticut, North Carolina, South Carolina, Georgia, Florida, Alabama, and Mississippi. Antibody titers ranged from 1:160 to greater than or equal to 1:40,960. On the basis of adsorption tests, the antibodies detected appeared to be specific to Borrelia spirochetes. Seropositive rodents in the eastern and southern United States, areas where human cases of Lyme borreliosis have been reported, indicate a widespread geographic distribution of B. burgdorferi or a closely related spirochete.
Enzyme immunoassay for detection of immunoglobulin G (IgG), IgM, and IgA antibodies against type 6B pneumococcal capsular polysaccharide and cell wall C polysaccharide in chinchilla serum
Authors: Koskela M , Harris M , Giebink G S
Published: January 2020

Conjugation of the capsular polysaccharides of Streptococcus pneumoniae to protein carriers has introduced a new generation of pneumococcal vaccines which may be efficacious in preventing pneumococcal otitis media during infancy. The chinchilla model has been used extensively for studying the pathogenesis of pneumococcal otitis media and for testing the efficacy of early pneumococcal capsular polysaccharide (PCP) vaccines, but immunologic studies in the chinchilla have been limited by the lack of antibodies against specific immunoglobulin isotypes. By using affinity-purified rabbit immunoglobulin G (IgG) anti-chinchilla IgG, IgM, and IgA, we developed a sensitive enzyme immunoassay that is highly specific for IgG, IgM, and IgA antibodies against type 6B PCP (anti-6B) and against C polysaccharide in chinchilla serum. Antibody titers increased in serum from five chinchillas immunized with a type 6B outer membrane protein complex vaccine. Increases of anti-6B IgG and IgM antibody titers were more striking than increases of anti-6B IgA or anti-C polysaccharide IgG, IgM, or IgA titers were.
Comparison of ribotyping and restriction enzyme analysis using pulsed-field gel electrophoresis for distinguishing Legionella pneumophila isolates obtained during a nosocomial outbreak
Authors: Schoonmaker D , Heimberger T , Birkhead G
Published: January 2020

Because of the ubiquity of Legionella isolates in aquatic habitats, epidemiologic evaluation of Legionella pneumophila strains is important in the investigation and subsequent control of nosocomial outbreaks of legionellosis. In this study, ribotyping and restriction enzyme analysis by pulsed-field gel electrophoresis (PFGE) were used to compare isolates of L. pneumophila obtained from patients and the environment during a nosocomial outbreak with unrelated control strains. Restriction enzyme analysis by PFGE resolved 14 different patterns among the L. pneumophila serogroup 1 and L. pneumophila serogroup 6 isolates involved in the study. Two of the patterns were observed in the three L. pneumophila serogroup 6 isolates from patients with confirmed nosocomial infections and environmental isolates from the potable water supply, which was, therefore, believed to be the source of the patients' infections. Three more patterns that were not present in isolates from patients with legionellosis were seen in isolates from the hospital environment, demonstrating the presence of multiple strains in the hospital environment. In the outbreak, one distinct pattern occurred among the L. pneumophila serogroup 1 isolates from patients with nosocomial infections, suggesting a common source; however, the source could not be determined. By comparison, ribotyping generated five patterns. However, some control strains of both L. pneumophila serogroups 1 and 6 possessed the same ribotypes as were present in the outbreak isolates. Both techniques were used successfully to subtype the isolates obtained during the investigation of the outbreak. Furthermore, restriction enzyme analysis by PFGE was useful for subdividing ribotypes and for distinguishing strains involved in the outbreak from epidemiologically unrelated strains.
DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology
Authors: Zhang Y , Mazurek G H , Cave M D , Eisenach K D , Pang Y , Murphy D T , Wallace R J
Published: January 2020

Mycobacterium tuberculosis isolates were studied by comparing large restriction fragment (LRF) patterns produced by digestion of chromosomal DNA with infrequent-cutting endonucleases and pulsed-field gel electrophoresis. Four cultures of H37Rv and 36 clinical isolates of M. tuberculosis were compared by using DraI, AsnI, XbaI, and SpeI. DraI and AsnI allowed easy visual separation of 18 of 21 epidemiologically unrelated strains. XbaI and SpeI allowed discrimination of all 21 unrelated strains, including the 3 strains inseparable with DraI and AsnI, but comparison of LRF patterns was more tedious because of overlapping fragments. A total of 26 isolates belonging to 10 clusters of related isolates were compared by pulsed-field gel electrophoresis, with all related isolates giving identical LRF patterns. These included multiple isolates from the same patient or the same family. The same grouping of clustered isolates was obtained when BamHI DNA digests were hybridized with two probes from the insertion sequence IS6110. Long-term laboratory passage of H37Rv produced minimal detectable changes in LRF patterns. LRF patterns are useful tools for epidemiologic studies of tuberculosis without the need for radioactive or specific DNA probes.
Enzyme-linked immunoelectrotransfer blot test for diagnosis of human hydatid disease
Authors: Verastegui M , Moro P , Guevara A , Rodriguez T , Miranda E , Gilman R H
Published: January 2020

Sera from 71 patients with surgically confirmed hydatid disease (which is caused by Echinococcus granulosus) were studied by an enzyme-linked immunoelectrotransfer blot (EITB) assay. Sera from patients either with other cestode infections or with another illness were used as controls. Results of the EITB test for hydatidosis were compared with those of the double-diffusion (DD5) test and an enzyme-linked immunosorbent assay (ELISA). In the EITB assay with bovine lyophilized hydatid fluid, three antigen bands of 8, 16, and 21 kDa were diagnostically important. The sensitivity of the assay by using these antigen bands was 80% for hepatic cysts, 56% for pulmonary cysts, and 56% for cysts located in multiple organs. In sera from controls, the specificity of the EITB assay was 100%. Cross-reactions to the 8-, 16-, and 21-kDa bands occurred, respectively, in 12, 4, and 4% of sera from patients with cysticercosis. No cross-reactions were noted in patients infected with Hymenolepis nana. The ELISA in which swine hydatid fluid was used as the antigen was as sensitive as the EITB test but was less specific (80%) and frequently cross-reacted with sera from patients with other cestode infections. The sensitivity of the DD5 test, which uses sheep hydatid fluid, was low (47%) , but its specificity was as high as that of the EITB assay. However, in patients with cysticercosis, cross-reactions were observed in 23% of sera tested. Despite the higher sensitivity found with the EITB assay, 23% (n = 5) of the serum samples that were positive by the DD5 test were not detected by the EITB assay. The EITB assay offers greater sensitivity and specificity than do the ELISA and the DD5 test. The highest proportion of hydatid cases is detected when the EITB and DD5 tests are run simultaneously.
Evaluation of the Autoscan Walkaway system for rapid identification and susceptibility testing of gram-negative bacilli
Authors: Kelly M T , Leicester C
Published: January 2020

We evaluated the performance of the Autoscan Walkaway (W/A) system (MicroScan, Sacramento, Calif.) in conjunction with the fluorometric Neg Combo panels for rapid identification and susceptibility testing of gram-negative bacilli. Fermentative and nonfermentative gram-negative bacilli were tested in parallel with the W/A system and the Cathra Repliscan replicator (C/R) system (Cathra, St. Paul, Minn.). Conventional biochemical testing and agar dilution testing were used to resolve the identification and susceptibility testing discrepancies. Of 495 clinical isolates tested, 445 (90%) were correctly identified by the W/A system and 483 (98%) were correctly identified by the C/R system. Repeat testing by using updated versions of the W/A system's computer identification software failed to demonstrate improved identification accuracy. For susceptibility testing, the W/A system demonstrated 5.6% total interpretative category errors, including only 0.9% major and very major errors. The comparative C/R system produced only 1% errors overall, including 0.2% major and very major errors. Although the W/A system is highly automated and is capable of producing results rapidly, our findings suggest that additional identification and susceptibility testing refinements are needed before the system will be suitable for routine use.
Epidemiological analysis of serologically determined rotavirus and enterotoxigenic Escherichia coli infections in Ecuadorian children
Authors: Brüssow H , Rahim H , Freire W
Published: January 2020

The statistical association of rotavirus- and enterotoxigenic Escherichia coli-specific serum antibody with demographic and hygienic factors was tested in Ecuadorian children enrolled in a cross-sectional survey. In 7- to 10-month-old children, enterotoxigenic E. coli-specific antibody was associated (P less than 0.05) with poor drinking water quality, lack of a sewage system, and feeding of supplementary food. In 7- to 14-month-old children, rotavirus-specific antibody was associated only with family size but notably not with hygienic factors.
Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii
Authors: Gouby A , Carles-Nurit M J , Bouziges N , Bourg G , Mesnard R , Bouvet P J
Published: January 2020

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.
Serotype variation of group A rotaviruses over nine winter epidemics in southeastern New England
Authors: Begue R E , Dennehy P H , Huang J , Martin P
Published: January 2020

By the use of enzyme immunoassay and RNA electrophoresis, rotavirus serotyping was performed with stool specimens from 605 children. Serotypes 1, 2, 3, and 4 accounted for 63, 9, 9, and 19% of the cases, respectively, with considerable yearly variation. An average of three serotypes cocirculated each year.
Detection of vancomycin resistance in Enterococcus species
Authors: Willey B M , Kreiswirth B N , Simor A E , Willaims G , Scriver S R , Phillips A , Low D E
Published: January 2020

Enterococcus faecalis and Enterococcus faecium isolates that are resistant to vancomycin have recently been identified in North America and Europe. Of 155 clinical isolates of enterococci (113 E. faecium and 42 E. faecalis), we found that 98 were resistant, 52 were moderately susceptible, and 5 had intermediate susceptibilities to vancomycin by using broth microdilution susceptibility testing according to the National Committee for Clinical Laboratory Standards (NCCLS) (Approved Standard M7-A2). Using NCCLS disk diffusion methodology (Approved Standard M2-A4), we evaluated the NCCLS supplemental M100-S3 revisions for zone diameter interpretive standards and incubation conditions and found 5.8% minor errors. A total of 234 isolates, which included an additional 79 E. faecium isolates that were moderately susceptible to vancomycin, were used to evaluate the Vitek GPS-TA card (bioMerieux, Inc., Hazelwood, Mo.) and the Pos MIC type 6 panel (MicroScan; Baxter Health Care Corp., West Sacramento, Calif.) for the detection of vancomycin resistance. The Vitek card was 100% specific and 72% sensitive, whereas the MicroScan panel with the Walk/Away system was 98% specific, with a sensitivity of 93% which increased to 99% when readings were performed manually. An agar screen plate method was evaluated with vancomycin concentrations of 6, 8, 10, or 12 micrograms/ml; plates were inoculated so as to obtain a final concentration of 10(5) CFU per spot. This method was found to be 100% sensitive and specific at all concentrations.
Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms
Authors: Goh S H , Byrne S K , Zhang J L , Chow A W
Published: January 2020

Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.
Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene
Authors: Brakstad O G , Aasbakk K , Maeland J A
Published: January 2020

Synthetic oligonucleotide primers of 21 and 24 bases, respectively, were used in the polymerase chain reaction (PCR) to amplify a sequence of the nuc gene, which encodes the thermostable nuclease of Staphylococcus aureus. A DNA fragment of approximately 270 bp was amplified from lysed S. aureus cells or isolated DNA. The PCR product was detected by agarose gel electrophoresis or Southern blot analysis by using a 33-mer internal nuc gene hybridization probe. With S. aureus cells the lower detection limit was less than 10 CFU, and with the isolated target the lower detection limit was 0.69 pg of DNA. The primers recognized 90 of 90 reference or clinical S. aureus strains. Amplification was not recorded when 80 strains representing 16 other staphylococcal species were tested or when 20 strains representing 9 different nonstaphylococcal species were tested. Some of the non-S. aureus staphylococci produced thermostable nucleases but were PCR negative. The PCR product was generated when in vitro-cultured S. aureus was used to prepare simulated clinical specimens of blood, urine, cerebrospinal fluid, or synovial fluid. No PCR product was generated when the sterile body fluids were tested. However, the sensitivity of the PCR was reduced when S. aureus in blood or urine was tested in comparison with that when bacteria in saline were tested. With the bacteria in blood, the detection limit of the PCR was 10(3) CFU. A positive PCR result was recorded when a limited number of clinical samples from wounds verified to be infected with S. aureus were tested, while the PCR product was not detected in materials from infections caused by other bacteria. Generation of PCR products was not affected by exposure of S. aureus to bactericidal agents, including cloxacillin and gentamicin, prior to testing, but was affected by exposure to UV radiation. The PCR for amplification of the nuc gene has potential for the rapid diagnosis of S. aureus infections by direct testing of clinical specimens, including specimens from patients with ongoing antimicrobial therapy.
Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses
Authors: Ojeh C K , Tsunemitsu H , Simkins R A , Saif L J
Published: January 2020

A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.
Role of coproantibody in clinical protection of children during reinfection with rotavirus
Authors: Coulson B S , Grimwood K , Hudson I L , Barnes G L , Bishop R F
Published: January 2020

Rotavirus is the major cause of severe, dehydrating infantile gastroenteritis. Infection is limited to the gut, but the relative roles of serum and secretory copro-immunoglobulin A (IgA) in protection are unclear. Specific copro-IgA is predictive of duodenal antirotaviral IgA and correlates with virus-neutralizing coproantibody. Copro-IgA conversion is a more sensitive marker of rotavirus reinfection than seroconversion. We measured rotavirus reinfections by copro-IgA conversion prospectively in 35 children recruited at a time of severe rotavirus illness. The children were followed up longitudinally for 14 to 31 months to determine whether high coproantibody levels correlated with clinical protection against rotavirus disease. Ninety-four percent of the children experienced reinfection, and 38% developed persistent elevations in specific copro-IgA termed plateaus. Plateau children had a higher mean annual rate of rotavirus infection and a lower ratio of symptomatic to total number of rotavirus reinfections than did nonplateau children. The annual rates of rotavirus infection and disease were significantly higher outside the plateau than inside it in children experiencing antirotavirus copro-IgA plateaus. Frequent rotavirus infection of children appears to stimulate production of a specific copro-IgA plateau which correlates with protection against an excess of infection and symptomatic disease.
Conserved sequences of the adenovirus genome for detection of all human adenovirus types by hybridization
Authors: Scott-Taylor T H , Hammond G W
Published: January 2020

The application of DNA hybridization directly to clinical specimens has the potential of improving the diagnosis of fastidious types of adenovirus. In this study, the genome of one adenovirus type from each human subgenus (A to F) was systematically evaluated by hybridization for homologous sequences to find the optimal common probe for detection of all human adenovirus types. The area of cross-hybridization, most closely defined with adenovirus type 2 (Ad2), mapped from map units 11.4 to 16.1 and 26.9 to 29.7 and, principally, to a central area of the genome between map units 47.5 and 65.2. The last area, enclosing the hexon gene, was highly conserved. Cloned probes generated from this area demonstrated the greatest homology to heterologous types by hybridization analysis. A HindIII-BglII clone containing the hexon gene of Ad2 within narrow confines reacted most evenly with all adenoviral types and detected the DNA of all other subgenera with a sensitivity 2 logs greater than that of a complete genomic Ad2 probe. The most homologous adenoviral gene sequences were observed in genes involved with DNA replication or intimately connected to the hexon in the early capsid formation. These results show that the hexon gene constitutes the best single region of the adenovirus genome for use as a genus-specific probe for the diagnosis of all human adenoviral subgenera by DNA hybridization.
Efficacy of an enzyme-linked immunosorbent assay for detection of urinary tract immunoglobulins for diagnosis of urinary tract infections
Authors: Kellogg J A , Manzella J P , Seiple J W , Fortna S J , Cook J W , Levisky J S
Published: January 2020

Results of the Uristat test (Shield Diagnostics Ltd.), a novel enzyme-linked immunosorbent assay (ELISA) for detection of urine antibodies to seven common bacterial pathogens, were compared with results of urine culture, urinalysis, and clinical history to determine the usefulness of Uristat in the diagnosis of urinary tract infections (UTIs). Midstream, catheterized, and indwelling catheter urine specimens sent to the laboratory for culture were included in the study. Quantitative cultures were performed on both 5% sheep blood agar and eosin-methylene blue agar. Uristat ELISAs were performed according to the manufacturer's instructions. By using a Bacillus subtilis bioassay technique, antibacterial activity was detected in the urine of 236 (22.2%) of 1,061 patients. Probable, possible, or asymptomatic UTIs were diagnosed for 258 (24.3%) of the 1,061 patients. Of those infections, 219 (84.9%) were caused by bacterial species whose antibodies were detectable by Uristat. Uristat's sensitivity and specificity were 76.7 and 56.0%, respectively. Uristat's predictive values of positive and negative results were 31.2 and 90.2%, respectively. Further development of the Uristat test is necessary before it can be of assistance in the diagnosis of UTIs.
Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products
Authors: Ubukata K , Nakagami S , Nitta A , Yamane A , Kawakami S , Sugiura M , Konno M
Published: January 2020

In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphatase-conjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from greater than 5 x 10(2) CFU per tube for Staphylococcus aureus to greater than 5 x 10(3) CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 microgram/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.
Ongoing group B Neisseria meningitidis epidemic in São Paulo, Brazil, due to increased prevalence of a single clone of the ET-5 complex
Authors: Sacchi C T , Pessoa L L , Ramos S R , Milagres L G , Camargo M C , Hidalgo N T , Melles C E , Caugant D A , Frasch C E
Published: January 2020

Beginning in 1988, the incidence of meningococcal disease in the area of greater São Paulo began to surpass the upper confidence limit of an 8-year average incidence (from 1979 to 1986), thus characterizing a new epidemic in the region of greater São Paulo. This epidemic, which extended to 1990, was different from previous epidemics in that it was caused by serogroup B. The increased incidence of meningococcal disease was paralleled by an increased prevalence of a single group B clone, B:4:P1.15, of the ET-5 complex. ET-5 strains have been present in the greater São Paulo area since 1979; however, they have been associated with a high percentage of the group B disease only from 1987 to the present. On the basis of the increased incidence of group B disease in São Paulo, a mass vaccination program with a serotype 4:P1.15 meningococcal protein vaccine was undertaken. The impact of this vaccination program is under analysis.
Immunoglobulin G antibodies to Helicobacter pylori in patients with dyspeptic symptoms investigated by the western immunoblot technique
Authors: Andersen L P , Espersen F
Published: January 2020

Helicobacter pylori is a gram-negative, curved, rod-shaped bacterium known to cause gastritis and to be an important factor in the pathogenesis of peptic ulcers. Serological testing has recently been proposed as an aid in diagnosis of H. pylori infections. In this study, we used the Western immunoblot technique to evaluate the possibility of using one or more of the antigens from H. pylori for this purpose. Thirteen major bands and about 30 minor bands could be identified by Western blotting when sera from 53 consecutive dyspeptic patients, 27 healthy children, and 25 blood donors were evaluated. Antibodies against most of the major bands were found significantly more frequently in patients with H. pylori infections than in patients without such infections. However, antibodies against a single polypeptide were not produced by all patients with H. pylori infection. Polypeptides with molecular masses of 120, 50, and between 19 and 36 kDa seem to be the most specific polypeptides for the diagnosis of H. pylori infections. This study showed only minor differences in antigenic composition between different clinical isolates of H. pylori, and serological cross-reactions with other bacteria were limited. Major serological cross-reactions were found only with Campylobacter jejuni and with bacterial lipopolysaccharide. However, one band at 60 kDa reacted with antiserum to the Legionella micdadei common antigen, which may indicate a cross-reaction with common antigen from several other bacteria. This study demonstrates that a number of bands may be useful as antigens in serological tests after isolation and purification.
Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California
Authors: Williams S G , Sacci J B , Schriefer M E , Andersen E M , Fujioka K K , Sorvillo F J , Barr A R , Azad A F
Published: January 2020

The recent discovery of cat fleas (Ctenocephalides felis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.
Secondary immune response in a vaccinated population during a large measles epidemic
Authors: Ozanne G , d'Halewyn M A
Published: January 2020

The rates of secondary immune response (SIR) and secondary vaccine failure (SVF) during a measles epidemic (10,184 notifications) were evaluated. A patient with SIR was defined as a subject for whom all sera were immunoglobulin G (IgG) positive and IgM negative with a significant increase in complement fixation titer. A patient with SVF was defined as a vaccinated symptomatic subject showing a SIR. Sequential sera from 898 subjects were tested for measles antibody by enzyme-linked immunosorbent assay (IgG and IgM) and by complement fixation. Evidence of recent anti-measles virus specific immune response was found in 496 subjects (55.5%). The vaccination rate was estimated at 74.6% (99% confidence interval [CI], 67.9 to 80.7%). The number of exposed vaccinated subjects was estimated at 370 (74.6% of 496). The SIR rate was 4.03% (20 of 496) (99% CI, 2.1 to 6.9%) among subjects with immune response. These 20 subjects were 2 with measles (Centers for Disease Control's definition), 6 with measles with rash of unknown duration, 8 with presumed measles with either rash or fever, 3 asymptomatic subjects (2 with recent contact with a measles case), and 1 undocumented subject. Since 3 patients with SIR were asymptomatic and 2 others were documented as not vaccinated, there was a maximum of 15 probable occurrences of SVF among the 20 patients with SIR. The SVF rate among exposed vaccinated subjects was estimated at 4.05% (15 of 370) (99% CI, 1.9 to 7.5%). In conclusion, neither prior vaccination nor detectable SIR ensures protective immunity. Measles virus may induce asymptomatic SIR in IgG-seropositive subjects. SVF led to typical or modified measles but did not seem to have played an important role during this epidemic.
Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome
Authors: Brian M J , Frosolono M , Murray B E , Miranda A , Lopez E L , Gomez H F , Cleary T G
Published: January 2020

Two pairs of oligonucleotide primers were designed to amplify fragments of the genes for Shiga-like toxin I (SLT-I) and SLT-II in a single reaction. A 370-bp segment and a 283-bp segment were amplified for SLT-I and SLT-II, respectively. The specificities of the polymerase chain reaction (PCR) amplification products were confirmed by using radioactively labeled oligonucleotide probes. SLT sequences were amplified from DNA isolated from 13 previously characterized enterohemorrhagic Escherichia coli (EHEC) strains. No amplification product was produced by using DNA from 20 non-EHEC strains. As little as one bacterial genome was detectable. PCR was then applied to DNA isolated directly from stool samples. We had to remove inhibitors of PCR that were present in lysates prepared from stool samples before amplification was achieved. First, we evaluated the sensitivity of PCR for the detection of known numbers of EHEC added to normal stools. Second, three children with SLT in their stools were shown to have SLT sequences in their stools by PCR. Two of these children had hemolytic-uremic syndrome, and a third child was asymptomatic. Stool specimens collected from another 26 asymptomatic children were negative by PCR for SLT sequences. PCR can be used to diagnose EHEC infections without prior culture of stool specimens.
rRNA-based method for sensitive detection of Babesia bigemina in bovine blood
Authors: Reddy G R , Dame J B
Published: January 2020

Three synthetic oligonucleotide probes complementary to unique regions of Babesia bigemina small-subunit rRNA were developed for detecting the parasite in bovine blood. These probes specifically detected a parasitemia of 2 x 10(-5)% by autoradiography in less than 24 h by using a 200-microliters sample of bovine blood. These probes did not bind to total RNA or genomic DNA isolated from another closely related species, Babesia bovis, or to bovine leukocyte RNA. This method detected B. bigemina infections in calves inoculated with as few as 1,000 infected erythrocytes from the second day onward for 16 days.
Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis
Authors: Plikaytis B B , Plikaytis B D , Yakrus M A , Butler W R , Woodley C L , Silcox V A , Shinnick T M
Published: January 2020

A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns.
Antibody responses to lipid A, core, and O sugars of the Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients
Authors: Kronborg G , Fomsgaard A , Galanos C , Freudenberg M A , Høiby N
Published: January 2020

Enzyme-linked immunosorbent assays were developed separately for the three main parts of the Pseudomonas aeruginosa lipopolysaccharide (LPS) molecule, namely, lipid A, core, and O polysaccharide. Anti-lipid A, anticore, and anti-O polysaccharide antibodies were measured in serum samples from 12 patients with cystic fibrosis (CF) in a longitudinal study covering the period before P. aeruginosa infection was established through at least 5 years of chronic infection. The serum antibody response to all parts of the P. aeruginosa LPS molecule increased during the course of chronic infection. The increase in anti-lipid A antibodies was specific for P. aeruginosa lipid A, since no increase in anti-Escherichia coli lipid A antibodies was seen. Immunoglobulin G, A, and M (IgG, IgA and IgM) antibodies were all involved in the specific systemic response to P. aeruginosa lipid A, core, and the O polysaccharides. IgG and IgA levels in particular increased during the course of infection and were significantly higher than the antibody increase seen with age in a healthy control group. The local immune response in the lungs was investigated by measuring IgG, IgA, and IgM antibodies to the separate parts of the P. aeruginosa LPS molecule in sputum samples from 18 CF patients with at least a 5-year history of chronic P. aeruginosa infection. Antibodies detected in sputum were mainly anti-lipid A and anti-O polysaccharide antibodies of the IgG and IgA isotypes. Very high IgA anti-lipid A titers were detected in sputum samples from some CF patients.
Isotype antibody response in cows to Streptococcus agalactiae group B polysaccharide-ovalbumin conjugate
Authors: Rainard P
Published: January 2020

Adult dairy cows were immunized with group B antigen (GBA) of Streptococcus agalactiae or GBA coupled to ovalbumin, both emulsified in incomplete Freund adjuvant, and their sera were examined by an enzyme-linked immunosorbent assay measuring bovine immunoglobulin isotypes (immunoglobulin G1 [IgG1], IgG2, and IgM) specific for GBA. All of the cows possessed naturally acquired antibodies against GBA, which implied that primary antibody responses could not be studied. At the highest dose tested (200 micrograms), free GBA elicited a slight increase in antibody titers only in the IgM isotype, to which most of the naturally acquired antibodies to GBA belonged. A second administration of antigen was not more effective. The conjugate was able to induce a strong humoral response against GBA, particularly in the IgG1 and IgG2 subisotypes, and a second injection of the conjugate induced a doubling of the peak antibody titers. Therefore, conjugation of GBA to a protein carrier markedly improved the antibody response, which showed the main characteristics of T-cell dependency. The opsonic activity of serum against an unencapsulated strain of S. agalactiae was reinforced by the immunization with the conjugate.
Comparison of a chemiluminometric immunoassay with culture for diagnosis of chlamydial infections in infants
Authors: Dumornay W , Roblin P M , Gelling M , Hammerschlag M R , Worku M
Published: January 2020

The performance of Magic Lite (CIBA-Corning), a chemiluminescent immunoassay (CIA), was compared with that of culture for the diagnosis of neonatal chlamydial conjunctivitis and respiratory infection. We performed a retrospective evaluation of 51 ocular and 96 nasopharyngeal specimens previously collected for culture testing with the CIA. The sensitivities for the ocular and the nasopharyngeal specimens were 91 and 91.7%, respectively. The specificities for both sites were 100%. A subsequent prospective study evaluating 71 ocular and 38 nasopharyngeal specimens revealed sensitivities of 83.3 and 20%, respectively. The specificities for both sites were 100%. The CIA performed favorably, compared with culture, for the diagnosis of chlamydial conjunctivitis; however, the CIA appeared less sensitive for the diagnosis of respiratory infection, including pneumonia.
Comparative evaluation of four commercially available monoclonal antibodies for culture confirmation of herpes simplex virus infection
Authors: Johnston S L , Wellens K
Published: January 2020

Four fluorescein isothiocyanate-labelled monoclonal antibody typing reagents were compared for intensity of fluorescence and sensitivity in confirming herpes simplex virus (HSV) in cell culture. A total of 125 (50 HSV type 1 [HSV-1] and 75 HSV-2) specimens positive for HSV were concurrently stained with type-specific monoclonal antibodies from Bartels (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), Kallestad (Pathfinder; Kallestad Diagnostics, Chaska, Minn.), Diagnostic Products Corp. (DPC) (PathoDx; DPC, Los Angeles, Calif.), and Syva (Microtrak; Syva Co., Palo Alto, Calif.). Fifty cultured specimens not displaying HSV-associated cytopathic effect were also stained. Each of the four reagents confirmed the 50 negative cultures and 125 positive cultures. One HSV-1-positive culture was missed by the Kallestad antibody. Ten HSV-1 isolates displayed dull fluorescence when stained with the Syva HSV-2 reagent. The other three reagents stained only the HSV-1 well with these 10 specimens. The Syva and Bartels stains were brighter, in general, than the Kallestad and DPC stains. However, the Kallestad and DPC stains were brilliant enough to allow interpretation without hesitation. The differences in intensity and background staining emphasize the need for laboratories to routinely perform quality checks on their reagents and to test new products which become commercially available.
Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients
Authors: Delgado R , Lumbreras C , Alba C , Pedraza M A , Otero J R , Gómez R , Moreno E , Noriega A R , Payá C V
Published: January 2020

The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.
Detection of rubella virus-specific polymeric immunoglobulin A by enzyme-linked immunosorbent assay in combination with streptococcal pretreatment of serum
Authors: Kawano K , Minamishima Y
Published: January 2020

An enzyme-linked immunosorbent assay combined with streptococcal treatment of serum was assessed for its ability to detect serum polymeric immunoglobulin A. This technique detects rubella virus-specific polymeric immunoglobulin A antibody, which appears for only a short time after infection, and it is useful for serodiagnosis of recent rubella virus infection.
Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification
Authors: Gupta B P , Jayasuryan N , Jameel S
Published: January 2020

The presence of hepatitis B virus DNA in the sera of individuals is the most definitive marker of an active viral infection. We have used polymerase chain reaction detection of hepatitis B virus DNA directly on whole blood dried as a spot on filter paper. The method is rapid, specific, and sensitive and has the ability to detect as little as 10 virus particles by ethidium bromide staining of the polymerase chain reaction-amplified products. The method is cost-effective, and the stability of the spots makes the collection and transportation of potentially infectious blood safe.