bluuebunny/crossref_metadata_2025 huggingface dataset:
Ability of canine Lyme disease vaccine to protect hamsters against infection with several isolates of Borrelia burgdorferi
Authors: Jobe D A , Callister S M , Lim L C , Lovrich S D , Schell R F
Published: January 2020

We used flow cytometry to determine levels of borreliacidal antibodies in hamsters after vaccination with a commercially available canine Lyme disease vaccine. In addition, we evaluated the ability of vaccinated hamsters to resist infection with several isolates of Borrelia burgdorferi. Borreliacidal antibodies could be detected 1 week after a primary vaccination, peaked at weeks 3 to 5, and then rapidly declined. One week after a booster vaccination, borreliacidal activity was detected at a dilution of 1:10,240, and it decreased fourfold by week 10 after the booster vaccination. Vaccinated hamsters were protected against infection with < or = 10(6) B. burgdorferi 297 organisms during the peak borreliacidal response (5 weeks after primary vaccination or 2 weeks after booster vaccination). However, hamsters were not fully protected from development of Lyme arthritis when the titer of borreliacidal antibodies was < 1:5,120. In addition, no significant borreliacidal activity was induced against B. burgdorferi C-1-11, LV4, or BV1, which belong to three other seroprotective groups. These studies demonstrate that vaccination with the canine Lyme disease vaccine induces protective antibodies against B. burgdorferi 297. However, significant levels of borreliacidal antibodies are not produced until 5 weeks after vaccination, and protection is short-lived. In addition, no borreliacidal activity was induced against other isolates of B. burgdorferi. Because of this, the incorporation of multiple isolates or protein subunits may be necessary to increase the effectiveness of future vaccines.
Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus
Authors: Cherian T , Bobo L , Steinhoff M C , Karron R A , Yolken R H
Published: January 2020

Influenza A virus infections are a major cause of morbidity and mortality worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivity. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcription PCR was performed with oligonucleotide primers directed at a highly conserved area of the influenza A matrix gene. Amplified DNA was identified by hybridization in solution to a nested biotinylated RNA probe and quantitated in an EIA. PCR-EIA detected small quantities of RNA from the three prevalent subtypes of human influenza A virus. Influenza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use in clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. Thirty-seven of the postinfection samples had detectable influenza A virus RNA by PCR-EIA, whereas only 26 postinfection samples were positive by culture. PCR-EIA was particularly efficient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were positive, whereas virus was cultivated from 4 samples (P < 0.00005). All preinfection samples from volunteers subsequently infected with influenza A virus were negative by PCR-EIA, as were samples from a volunteer infected with parainfluenza virus type 3. Nucleic acid amplification techniques represent important tools for the timely and sensitive diagnosis of influenza A virus infections and, therefore, their management and control.
Plague pandemics investigated by ribotyping of Yersinia pestis strains
Authors: Guiyoule A , Grimont F , Iteman I , Grimont P A , Lefèvre M , Carniel E
Published: January 2020

Yersinia pestis is the causative agent of plague, a disease which has caused the deaths of millions of people and which persists now in endemic foci. The rRNA gene restriction patterns (i.e., ribotypes) of 70 strains of Y. pestis, isolated on the five continents over a period of 72 years, were determined by hybridization with a 16S-23S rRNA probe from Escherichia coli. The combination of the EcoRI and EcoRV patterns resulted in the elucidation of 16 ribotypes. Two of them (B and O) characterized 65.7% of the strains studied, while the 14 other ribotypes were found in no more than three strains each. A relationship was established between biovars and ribotypes: strains of biovar Orientalis were of ribotypes A to G, those of biovar Antiqua were of ribotypes F to O, and those of biovar Medievalis were of ribotypes O and P. Great heterogeneity in rRNA restriction patterns was found among strains isolated in Africa; this heterogeneity was less pronounced among Asian isolates and was completely absent from the American strains. Pulsed-field gel electrophoresis was performed on the DNAs of some strains, but it appeared that different colonies from the same strain displayed different pulsed-field gel electrophoresis patterns and therefore that this technique was not suitable for comparison of Y. pestis isolates. In contrast, the ribotypes of individual colonies within a given strain were stable and were not modified after five passages in vivo. A clear correlation between the history of the three plague pandemics and the ribotypes of the strains could be established.
Inhibitory effect of the Isolator blood culture system on growth of Mycobacterium avium-M. intracellulare in BACTEC 12B bottles
Authors: Wasilauskas B , Morrell R
Published: January 2020

The examination of 6,938 clinical specimens collected during the period January 1991 through December 1992 suggested that the Isolator blood culture system (Wampole) inhibited growth of Mycobacterium avium-M. intracellulare complex (MAC) in BACTEC 12B medium. Of 162 MAC blood culture isolates, 94% were recovered from Lowenstein-Jensen (LJ) medium, while only 50% were recovered from 12B medium. The time to detection with LJ medium was 18 days, while that with 12B medium was 24 days. In contrast, 62% of the 305 MAC nonblood culture isolates were recovered from the LJ medium, while 87% were found in the 12B medium. The time to detection for these cultures was also reversed, i.e., 28 days for LJ medium versus 15 days for 12B medium. Dilution studies using the lysis-anticoagulant reagent from Isolator tubes demonstrated inhibition of both clinical and American Type Culture Collection strains of MAC, even at low concentrations of lysis-anticoagulant reagent. Washing the Isolator blood sediment prior to inoculating the 12B bottles eliminated any growth inhibition. Clinical and experimental data suggest that the use of the Isolator blood culture tube with the BACTEC 12B medium is contraindicated for mycobacterial blood cultures.
Detection of Klebsiella pneumoniae and Escherichia coli strains producing extended-spectrum beta-lactamases
Authors: Katsanis G P , Spargo J , Ferraro M J , Sutton L , Jacoby G A
Published: January 2020

Plasmids encoding extended-spectrum beta-lactamases of the TEM, SHV, and AmpC families were introduced into common Escherichia coli and Klebsiella pneumoniae hosts to create a homogeneous panel for evaluating the abilities of five test systems to detect resistance to eight beta-lactam antibiotics. Although MICs, as determined by agar dilution or E test strips, were increased and disk diffusion zone diameters were diminished, breakpoints for resistance were often not reached, and neither approach was sensitive in detecting resistance to oxyimino-beta-lactams. The MicroScan 18-h microdilution or Vitek rapid automated procedures were similarly insensitive. Ceftazidime was the best single test antibiotic for detecting extended-spectrum beta-lactamase production. beta-Lactamases TEM-7 and TEM-12 were particularly difficult to detect. Because of such difficulties, the prevalence of extended-spectrum beta-lactamases is likely to be greater than is currently appreciated.
Evaluation of enzyme immunoassay for Candida cytoplasmic antigens in neutropenic cancer patients
Authors: Morhart M , Rennie R , Ziola B , Bow E , Louie T J
Published: January 2020

A Candida albicans cytoplasmic antigen enzyme immunoassay (CACP antigen EIA) was developed with antibodies raised against antigens prepared from yeast cells grown under standardized growth conditions. The C. albicans components reactive in the EIA were shown to be predominantly proteins with associated carbohydrates. Denaturing gel electrophoresis revealed the presence of five major CACP proteins with molecular weights between 36,000 and 44,000. The clinical usefulness of the CACP EIA was evaluated by retrospective blinded measurement of 89 serum samples from 31 granulocytopenic patient episodes. Twice-weekly surveillance cultures, sequential serum samples (approximately once per week or with change of the clinical course), and standard diagnostic criteria of fungal infection were used to categorize patients. The sensitivity and specificity of the CACP assay on the basis of serum samples were 82 and 100%, respectively (67 and 100% on the basis of patient episodes). The positive and negative predictive values were 100 and 97% for serum (100 and 93% for patient episodes). By comparison, the CANDTEC assay had low sensitivity (33%) and poor positive predictive values (50%). The CACP EIA may be a useful test suitable for further evaluations as a method for the diagnosis of invasive Candida infection in neutropenic cancer patients.
Sequence variation in Shigella sonnei (Sonnei), a pathogenic clone of Escherichia coli, over four continents and 41 years
Authors: Karaolis D K , Lan R , Reeves P R
Published: January 2020

The genetic variation within a bacterial clone (known as Shigella sonnei but in effect a clone of Escherichia coli) was examined by studying 46 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over a period of 41 years (1950 to 1991). Restriction fragment length polymorphism and DNA sequencing of two housekeeping genes, mglB and gnd, in 10 strains revealed only one nucleotide substitution in the mglB gene in one strain. Ribotyping of 31 strains recovered from five countries over the 41 years proved more sensitive, detecting eight polymorphic sites with worldwide change in frequency of alleles at one site over the period studied.
Differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of PCR-amplified DNA
Authors: Eremeeva M , Yu X , Raoult D
Published: January 2020

Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified genes was used to study spotted fever group (SFG) rickettsiae, extending the previous work of Regnery et al. (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991). Twenty-six strains of SFG rickettsia were studied, including several recognized species which have never been studied (R. parkeri, R. helvetica, and R. japonica) as well as strains which are not currently classified. Two previously used primer pairs derived from the R. prowazekii citrate syntase gene and the R. rickettsii 190-kDa protein antigen gene were studied, as were primer pairs obtained from the R. rickettsii 120-kDa protein antigen gene. By using three amplifications and three enzyme digestions, it was possible to differentiate between almost all of the known SFG rickettsia species and to differentiate between several strains of the R. conorii complex. Two human pathogens, "R. africae" and the Israeli tick typhus rickettsia, were first separated by using BG-12 pair primer amplification and then RsaI restriction endonuclease digestion. The proposed simplified model of identification may be useful in studying the geographical distributions of SFG rickettsiae.
Evaluation of an automated immunodiagnostic assay, VIDAS Rotavirus, for detection of rotavirus in fecal specimens
Authors: Dennehy P H , Schutzbank T E , Thorne G M
Published: January 2020

Four hundred sixty-four fecal specimens were tested for rotavirus by three immunoassays, VIDAS Rotavirus (bioMérieux Vitek, Hazelwood, Mo.), Rotaclone (Cambridge Biotech Corporation, Worcester, Mass.), and Pathfinder Rotavirus (Sansfi Diagnostics Pasteur, Chaska, Minn.). Twenty-seven discordant specimens were evaluated by electron microscopy. The sensitivities, specificities, positive predictive values, and negative predictive values were 98, 99.3, 98.7, and 99%, respectively, for VIDAS Rotavirus; 100, 99, 98.1, and 100%, respectively, for Rotaclone; and 100, 92.4, 87.3, and 100%, respectively for Pathfinder Rotavirus.
Interference of immunoglobulin G (IgG) antibodies in IgA antibody determinations of Chlamydia pneumoniae by microimmunofluorescence test
Authors: Jauhiainen T , Tuomi T , Leinonen M , Kark J D , Saikku P
Published: January 2020

In the microimmunofluorescence test for measuring immunoglobulin A (IgA) antibodies against Chlamydia pneumoniae, removal of interfering IgG antibodies made IgA antibody reactivity patterns in 952 serum samples easier to interpret, prozone effects disappeared, and titers increased, especially in the sera with high IgG titers. IgA rheumatoid factors did not interfere in the assay.
Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism
Authors: Frénay H M , Theelen J P , Schouls L M , Vandenbroucke-Grauls C M , Verhoef J , van Leeuwen W J , Mooi F R
Published: January 2020

The X region of the protein A gene of Staphylococcus aureus contains a highly polymorphic sequence which is composed of repeats of 24 bp. We used amplification by PCR to investigate whether this region could be used to discriminate between epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains. Most epidemic MRSA strains (24 of 33) harbored more than seven repeats, while most nonepidemic MRSA strains (10 of 14) contained seven or fewer repeats. It is conceivable that a longer X region results in a better exposition of the Fc-binding region of protein A, thereby facilitating colonization of host surfaces and contributing to the epidemic phenotype.
Evaluation of MicroScan rapid gram-positive panels for detection of oxacillin-resistant staphylococci
Authors: Woods G L , LaTemple D , Cruz C
Published: January 2020

The ability of MicroScan rapid panels (Baxter MicroScan, West Sacramento, Calif.) to detect oxacillin resistance in 92 clinical isolates of Staphylococcus aureus and 103 coagulase-negative staphylococci was evaluated by comparing results with those of MicroScan 24-h MIC panels and, to resolve discrepancies, oxacillin agar screening. Both panels were interpreted by the MicroScan WalkAway-96 system. Rapid panels detected 96.7% of resistant S. aureus isolates and 72% of resistant coagulase-negative staphylococci, 22 of which did not grow in the panels.
Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources
Authors: Gautom R K , Lory S , Seyedirashti S , Bergeron D L , Fritsche T R
Published: January 2020

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA fingerprinting) was evaluated as an epidemiologic tool for identifying potential reservoirs of Acanthamoeba infection. Fingerprints for 15 clinical isolates recovered by our affiliated laboratories were compared with those for 25 environmental isolates from western Washington State and 10 American Type Culture Collection (ATCC) strains. Seven different fingerprint groups emerged from the analysis of clinical isolates with six selected restriction enzymes (BamHI, BglII, EcoRI, HindIII, KpnI, and SalI). Fourteen (56%) environmental and 4 (40%) ATCC isolates displayed fingerprints similar to those of clinical isolates. In all, five of the seven groups contained one or more environmental and/or ATCC isolates. Comparisons with published mtDNA fingerprints for Acanthamoeba isolates showed that two groups have counterparts in Europe and Japan and in Europe and Australia. The inclusion of environmental isolates demonstrated that the most common clinical isolates do have counterparts readily recoverable from the surrounding environment and that some of these counterparts appear to be geographically widespread.
Distribution of serotypes and antimicrobial resistance of Streptococcus pneumoniae strains isolated in Brazil from 1988 to 1992
Authors: Sessegolo J F , Levin A S , Levy C E , Asensi M , Facklam R R , Teixeira L M
Published: January 2020

Forty-two serotypes were identified among 288 Streptococcus pneumoniae strains isolated from patients living in Brazil. Serotyping was determined by the capsular typing test (Quellung reaction). Types 14 (10.4%), 6B (9.8%), 23F (8.0%), 5 (7.3%), 19F (6.9%), 6A (6.0%), and 1 and 4 (4.6%) were the most commonly identified strains. Two hundred twenty (76.4%) of the strains were of serotypes that are included in the 23-valent pneumococcal polysaccharide vaccine. If vaccine-related serotypes are also considered, the proportions of coverage in the vaccine are 82.3% (if type 6B alone is added) and 85.7% (if all the vaccine-related types are considered to be cross-protecting). Decreased susceptibility to penicillin, which was identified by using the 1-microgram oxacillin disk method as a screening test, was detected in 70 (26.7%) strains. The MICs of nine antimicrobial agents were determined by using the procedures recommended by the National Committee for Clinical Laboratory Standards. Seventy (35.9%) of the strains were resistant to tetracycline, 57 (29.2%) were resistant to sulfamethoxazole-trimethoprim, 3 (1.5%) were resistant to rifampin, 2 (0.80%) were resistant to penicillin, and 1 (0.5%) was resistant to chloramphenicol. The two penicillin-resistant strains were also resistant to or had decreased susceptibilities to cephalosporins. Forty-seven (17.9%) of the strains were intermediately resistant to penicillin, 17 (8.7%) were intermediately resistant to tetracycline, 13 (6.7%) were intermediately resistant to chloramphenicol, 12 (6.1%) were intermediately resistant to erythromycin, and 6 (3.1%) were intermediately resistant to rifampin.
Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping
Authors: Johnson W M , Tyler S D , Rozee K R
Published: January 2020

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.
Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis
Authors: Maurin M , Roux V , Stein A , Ferrier F , Viraben R , Raoult D
Published: January 2020

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.
Serological detection of Helicobacter pylori antibodies in children and their parents
Authors: Best L M , Veldhuyzen van Zanten S J , Sherman P M , Bezanson G S
Published: January 2020

The antibody response to Helicobacter pylori was examined in 56 children (ages 5 to 18) to determine whether serological tests can be used for diagnosis. Twenty-four children (43%) were H. pylori positive and 32 children (57%) were H. pylori negative by culture and histological examination of endoscopic biopsy specimens. The immune response was also examined in 39 nonendoscoped parents of the children. H. pylori-specific immunoglobulin G (IgG) and IgA antibodies were detected by the flow microsphere immunofluorescent assay (FMIA). IgG was also detected by using the Pyloristat enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, and positive and negative predictive values for the FMIA for IgG were 100, 97, 96, and 100%, respectively. The respective values for the Pyloristat ELISA for IgG were 96, 94, 92, and 97%. The respective values for the FMIA for IgA were 50, 100, 100, and 73%. Both assays identified the same 19 parents as IgG positive, while FMIA identified 17 of the 19 parents as IgA positive.
Cellular fatty acid composition and phenotypic and cultural characterization of CDC fermentative coryneform groups 3 and 5
Authors: Bernard K , Bellefeuille M , Hollis D G , Daneshvar M I , Moss C W
Published: January 2020

Seventy strains of fermentative, asporogenous, gram-positive coccobacilli or short rods form two closely related groups which have been designated CDC fermentative coryneform groups 3 (32 strains, xylose fermenters) and 5 (38 strains, xylose nonfermenters). The two taxa are otherwise similar to each other phenotypically and culturally and by a distinctive Staphylococcus-like odor and by cellular fatty acid (CFA) composition. CDC group 3 and CDC group 5 strains have been isolated from clinical sources (blood, abscesses, and wounds but not urine or respiratory specimens) in Canada and the United States and among referrals from Belgium, Sweden, and Spain. Coryneform CDC group 3 strains were phenotypically similar to CDC coryneform group A-3 but were distinguishable by their inability to reduce nitrate and by their lack of motility. Coryneform CDC group 5 isolates were phenotypically somewhat similar to Actinomyces viscosus and Rothia dentocariosa, except that none of this group reduced nitrate. Both CDC groups could be differentiated from these similar bacteria by the ability to decarboxylate lysine and ornithine. The CFA compositions of CDC group 3 and 5 strains were similar to each other, were distinctive from those of other coryneforms, and were of the branched-chain type. API CORYNE codes were consistent for both CDC group 3 and CDC group 5 bacteria, suggesting that this method could be useful as an identification method.
Restricted association between biotypes and serotypes within group A streptococci
Authors: Bouvet A , Geslin P , Kriz-Kuzemenska P , Blanc V , Devine C , Grimont F
Published: January 2020

Investigating individual variations between different isolates of group A streptococci, we observed a close correlation between biotypes and serotypes in 46 strains from pharyngitis patients. Biotyping, carried out with a commercially available rapid identification gallery, delineated 10 different associations of characteristics, designated biotypes 1 to 10, observed both in the manufacturer's (127 strains) and our personal (98 strains) collections of group A strains. Only the most frequent biotypes (biotypes 1 to 6) were observed in the pharyngitis cohort, but the overall frequencies of the biotypes did not display striking differences compared with the control collections. Serotyping of the pharyngitis strains showed that each M type was restricted to a sole biotype. For example, M types 1, 4, and 28 were found only in biotype 1 and M type 6 was found only in biotype 6 strains. This association was not due to an epidemiologic bias, since it was also observed in a control series consisting of reference strains and isolates from distant countries (the United States and Czech Republic versus France). An exception was for M type 78, which exhibited biotype 3 or biotype 4. Investigation of the heterogeneity of the strains at the DNA level showed no significant variations of the ribotype patterns between strains of different biotypes, confirming that group A streptococci belong to a unique and homogeneous species. This previously undescribed association between serotypes and biotypes is of interest for a rapid and preliminary characterization of strains isolated in individual patients or during an outbreak. A possible pathogenic association of some biotypic characteristics with specific M proteins is envisaged.
Evaluation of rapid test for detection of antibody to human immunodeficiency virus type 1 and type 2
Authors: Asihene P J , Kline R L , Moss M W , Carella A V , Quinn T C
Published: January 2020

The Genie HIV-1/HIV-2 rapid assay was compared with an enzyme-linked immunosorbent assay and Western blot (immunoblot) by using 540 serum specimens from four different populations. The Genie HIV-1/HIV-2 assay was easy to perform, required no equipment, provided visual results within 10 min, and demonstrated excellent sensitivity and specificity compared with the Western blot.
Comparison of fecal lactoferrin latex agglutination assay and methylene blue microscopy for detection of fecal leukocytes in Clostridium difficile-associated disease
Authors: Yong W H , Mattia A R , Ferraro M J
Published: January 2020

The fecal lactoferrin assay was more sensitive (75%) than methylene blue microscopy (40%) for the detection of leukocytes in Clostridium difficile toxin-positive fecal samples. Although limited sensitivity and specificity precludes its use as a laboratory screening test, it may be a more useful initial test in an algorithm for clinically suspected C. difficile-associated disease.
Comparison of three rapid methods, tributyrine, 4-methylumbelliferyl butyrate, and indoxyl acetate, for rapid identification of Moraxella catarrhalis
Authors: Speeleveld E , Fossépré J M , Gordts B , Van Landuyt H W
Published: January 2020

Moraxella catarrhalis can easily be differentiated from other oxidase-positive, gram-negative cocci with tributyrine, 4-methylumbelliferyl butyrate, or indoxyl acetate. All M. catarrhalis give positive reactions, and all Neisseria spp. give negative reactions. The 4-methylumbelliferyl butyrate tube test and indoxyl acetate strip test provide same-day identification of M. catarrhalis isolates.
Infections with intestinal parasites in Peace Corps volunteers in Guatemala
Authors: Herwaldt B L , de Arroyave K R , Wahlquist S P , du Pée L J , Eng T R , Juranek D D
Published: January 2020

To assess the role of parasites in causing diarrhea in Peace Corps volunteers in Guatemala, 115 stool specimens from a case-control investigation (48 case [diarrhea] and 26 control episodes) were examined. A potentially pathogenic protozoan that could account for diarrheal illness was found for only 12% of the case episodes.
Isolation of Mycoplasma species from bronchoalveolar lavages of patients positive and negative for human immunodeficiency virus
Authors: Teel L D , Finelli M R , Johnson S C
Published: January 2020

The rates of isolation of Mycoplasma species from bronchoalveolar lavages of human immunodeficiency virus (HIV)-infected patients and HIV-negative patients were compared. Mycoplasma species were more frequently isolated from HIV-positive patients. In most cases, a known pulmonary pathogen was also identified. All samples tested negative for Mycoplasma fermentans by PCR.
Rotavirus serotype G5 associated with diarrhea in Brazilian children
Authors: Gouvea V , de Castro L , Timenetsky M C , Greenberg H , Santos N
Published: January 2020

Rotavirus serotype G5 in fecal specimens of 38 Brazilian children with diarrhea was identified by PCR and enzyme immunoassays. The strains exhibited long RNA electropherotypes and either subgroup II or nonsubgroup I-nonsubgroup II specificities. Serotype G5 has been found in piglets and horses but not yet in humans.
Evaluation of specificity of indirect enzyme-linked immunosorbent assay for diagnosis of human Q fever
Authors: Uhaa I J , Fishbein D B , Olson J G , Rives C C , Waag D M , Williams J C
Published: January 2020

Ninety-five acute- and convalescent-phase serum specimens from 48 patients suspected of having rickettsial or Legionella infections were assayed for antibodies to Coxiella burnetii, the causative agent of Q fever. To evaluate the specificity of the indirect enzyme-linked immunosorbent assay (ELISA) for human Q fever, we compared the ELISA results with those of the indirect immunofluorescence antibody (IFA) test. The ELISA data were analyzed by two different criteria for a positive test. The first criterion for positive results by ELISA was based upon diagnostic titers established in a study of 150 subjects who had no demonstrable cellular or humoral immune responses to C. burnetii phase I or phase II whole cells or phase I lipopolysaccharide. The second criterion was based upon diagnostic antibody titers in a study of 51 subjects who had been diagnosed as having clinical Q fever and had fourfold or greater rises in humoral immune responses to C. burnetii phase I and phase II whole-cell antigens. A comparison of the ELISA and IFA test results of the 95 serum specimens indicated excellent agreement between the tests (Kappa = 92.9%; P < 0.05). None of the 38 patients whose etiologies were confirmed serologically as Legionnaires' disease or rickettsial diseases other than Q fever were classified as positive for C. burnetii by the ELISA. Only one patient identified by the IFA test as having Q fever was not scored positive by the ELISA. These results suggest that the ELISA is useful for epidemiologic screening and as a diagnostic test for human Q fever.
Comparison of agar dilution, broth dilution, and disk diffusion testing of ampicillin against Haemophilus species by using in-house and commercially prepared media
Authors: Kohner P C , Rosenblatt J E , Cockerill F R
Published: January 2020

An evaluation to determine the optimal method for the in vitro susceptibility testing of Haemophilus strains to ampicillin was undertaken. In our hands, in-house-prepared Haemophilus Test Medium used by either the broth macrodilution or agar dilution method produced the most consistent results, especially for beta-lactamase-negative, ampicillin-resistant H. influenzae strains.
Evaluation of three commercially available blood culture systems for cultivation of Helicobacter pylori
Authors: Kehler E G , Midkiff B R , Westblom T U
Published: January 2020

Helicobacter pylori is unable to grow in regular blood culture systems, including the BACTEC (Johnston Laboratories), Septi-Chek (Hoffman-La Roche), and Bacto (Difco) systems. We tested three blood culture systems used for fastidious organisms: brucella broth with SPS and CO2 (Becton Dickinson), biphasic brain heart infusion agar or broth (Becton Dickinson), and supplemented peptone broth (Vacutainer). Blood culture bottles were inoculated with H. pylori and human blood and were then incubated by routine diagnostic laboratory procedures. All three blood culture systems were able to sustain the growth of H. pylori, but brucella broth had the highest CFU per milliliter after 72 h. We conclude that a diagnostic laboratory should be able to detect H. pylori bacteremia in a majority of cases by using brucella blood culture bottles.
Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus
Authors: Eiden J J , Mouzinho A , Lindsay D A , Glass R I , Fang Z Y , Taylor J L
Published: January 2020

Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated.
Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay
Authors: Wolfhagen M J , Fluit A C , Torensma R , Poppelier M J , Verhoef J
Published: January 2020

Rapid detection of toxigenic Clostridium difficile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples.
Development of enzyme-labeled oligonucleotide probe for detection of mecA gene in methicillin-resistant Staphylococcus aureus
Authors: Shimaoka M , Yoh M , Segawa A , Takarada Y , Yamamoto K , Honda T
Published: January 2020

A DNA hybridization method with an enzyme-labeled oligonucleotide probe (mecA-ELONP) was developed to detect the methicillin-resistant gene (mecA) in methicillin-resistant Staphylococcus aureus. For rapid identification, bacterial colonies were transferred from agar plates directly onto nylon membranes. Lysis of cells, denaturation of DNA, and hybridization were performed on the membranes. These procedures required only 3 h for completion. The results obtained by this test closely corresponded with those obtained by determining the MICs of oxacillin against S. aureus. The results of the mecA-ELONP also correlated well with those of a commercially available PCR test. Thus, mecA-ELONP proved to be a reliable and convenient method for the rapid identification of methicillin-resistant S. aureus, which could be useful in clinical microbiology laboratories.
Differentiation between human immunodeficiency virus type 1 (HIV-1) and HIV-2 isolates by nonradioisotopic reverse transcriptase-typing assay
Authors: Urabe T , Sano K , Nakano T , Odawara F , Lee M H , Otake T , Okubo S , Hayami M , Misaki H , Baba M
Published: January 2020

We tested whether human immunodeficiency virus type 1 (HIV-1) could be differentiated from HIV-2 by a reverse transcriptase (RT)-typing assay that measured the reduction of enzyme activity owing to specific antibody. RT-inhibiting antibody was examined for HIV type specificity by a new nonradioisotopic RT assay. Antibodies from four rabbits immunized with recombinant HIV-1 RT and from 23 HIV-1-seropositive individuals all specifically inhibited the enzyme activities of two HIV-1 strains (LAV-1 and GH-3), three zidovudine-resistant HIV-1 mutants, and a recombinant HIV-1 RT. However, none of these antisera affected the activities of six HIV-2 strains (GH-1, GH-2, GH-4, GH-5, GH-6, LAV-2ROD), Rous-associated virus type 2, and DNA polymerase I from Escherichia coli. In contrast, HIV-2 antibody from a rabbit immunized with disrupted GH-1 virions blocked the enzyme activities of the six HIV-2 strains but not those of the three HIV-1 strains, Rous-associated virus type 2, or DNA polymerase I. These results indicate that the antigenic domains of HIV-1 and HIV-2 RTs recognized by their inhibiting antibodies are distinct from each other and are highly conserved. Clinical HIV isolates from 18 HIV-1-seropositive individuals and 3 HIV-2-seropositive Ghanaian individuals were identified as HIV-1 and HIV-2, respectively, by the nonradioisotopic RT-typing assay.
Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique
Authors: Kessler H H , Pierer K , Weber B , Sakrauski A , Santner B , Stuenzner D , Gergely E , Marth E
Published: January 2020

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.
Detection of Brucella melitensis by BACTEC NR660 blood culture system
Authors: Yagupsky P
Published: January 2020

Data on the performance of modern blood culture systems for the detection of Brucella spp. are insufficient. To evaluate the performance of the BACTEC NR660 blood culture system for the detection of Brucella melitensis within the routine 1-week blood culture protocol, a prospective 24-month study was conducted in an area endemic for B. melitensis in southern Israel. Blood samples obtained from patients with suspected brucellosis were monitored and blindly subcultured once per week for 4 weeks, and the fraction of blood cultures positive for B. melitensis detected by the BACTEC NR660 instrument within the first week was determined. During the study period, a total of 373 blood cultures were obtained from patients in whom brucellosis was suspected, and 27 (7.2%) of them, drawn from 21 different patients, were positive for B. melitensis. Twenty-one (78.8%) of these positive cultures were detected by the BACTEC instrument within 7 days, and six positive cultures were detected by subculture after 2 or 3 weeks of incubation. It is concluded that the BACTEC NR660 blood culture system detects the majority of B. melitensis isolates within the routine 1-week blood culture schedule. To maximize the recovery of the organism, however, prolonged incubation and periodic performance of blind subcultures are still required.
Detection of cilia-associated respiratory bacillus by PCR
Authors: Cundiff D D , Besch-Williford C , Hook R R , Franklin C L , Riley L K
Published: January 2020

The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, motile bacterium that has been implicated as an etiologic agent of respiratory disease in laboratory rodents. In the present study, approximately 1,200 bases of the 16S rRNA gene from three CAR bacillus isolates were sequenced. CAR bacillus-specific primers were designed on the basis of the 16S rRNA gene sequence and used in a PCR assay. The PCR assay detected as little as 500 fg of purified CAR bacillus DNA. The expected 267-bp DNA fragment was amplified from respiratory tissue of frozen, formalin-fixed, and paraffin-embedded samples from experimentally and naturally infected rats and mice. In contrast, no product was amplified from respiratory tissues of sham-infected experimental animals or animals that were serologically or histopathologically negative for the CAR bacillus. Our findings indicate that this PCR assay is a rapid, specific, and sensitive detection method for the diagnosis of CAR bacillus infection in rats and mice.
Typing of group A streptococci by random amplified polymorphic DNA analysis
Authors: Seppälä H , He Q , Osterblad M , Huovinen P
Published: January 2020

Random amplified polymorphic DNA (RAPD) analysis was evaluated in comparison with restriction endonuclease analysis (REA) of genomic DNA and serotyping in the typing of 160 epidemiologically unrelated group A streptococci (GAS). Amplification of genomic DNA of GAS was performed with a single primer with an arbitrarily selected nucleotide sequence of 12 nucleotides. In total, 31 RAPD patterns and 15 REA patterns were observed among the isolates studied. The results of RAPD analysis were in accordance with the results of REA for 86% of the isolates, as both methods identified 15 different strains among 138 isolates. However, RAPD analysis differentiated 16 additional strains among 22 isolates. RAPD analysis was somewhat better than REA for differentiation of isolates of the same and different serotypes. However, not all of the serotypes were differentiated by RAPD analysis either. In conclusion, RAPD analysis provides a practical alternative for genomic typing of GAS. It can be recommended for the typing of GAS, especially if used in parallel with serotyping.
Comparison of four immunoserologic assays for detection of antibodies to Borrelia burgdorferi in patients with culture-positive erythema migrans
Authors: Mitchell P D , Reed K D , Aspeslet T L , Vandermause M F , Melski J W
Published: January 2020

In view of the significant sequelae associated with Lyme borreliosis, there is a need for timely and accurate diagnosis of erythema migrans (EM). Although Borrelia burgdorferi can be cultured from biopsies of EM lesions, immunodiagnostic testing is more widely available. Four immunoserologic methods were studied by using the sera of 51 patients with EM lesions that were culture positive for B. burgdorferi. Nineteen patients had single primary lesions, and thirty-two had multiple secondary lesions. At the time of biopsy, 40 patients, 8 with primary lesions and all patients with secondary lesions, were seropositive by an immunoglobulin M (IgM) indirect fluorescent-antibody (IgM IFA) test (Bion Enterprises). Twenty-three patients were seropositive by a whole-cell fluorescence enzyme immunoassay (EIA) (BioWhittaker, Inc.), twenty-two were positive by immunoblotting (ViroStat, Inc.), and one was positive by a P39 recombinant EIA (P39 EIA) (General Biometrics, Inc.). Sera from various patient control groups were tested: rheumatoid arthritis (n = 19), infectious mononucleosis (n = 20), systemic lupus (n = 22), syphilis (n = 13), streptococcal sequelae (n = 20), and healthy subjects (n = 16). None of these sera reacted with the IgM IFA test or P39 EIA. Fifteen reacted with the fluorescence EIA. We conclude that the IgM IFA test is an effective and reliable assay for the diagnosis of EM, particularly for patients with secondary lesions. Immunoblot, fluorescence EIA, and P39 EIA lack the sensitivity to reliably diagnose EM.
Clear broth and plate media for culture of Haemophilus ducreyi
Authors: Totten P A , Stamm W E
Published: January 2020

Using catalase as a source of heme, we have developed both clear plate and broth media for the growth of Haemophilus ducreyi, the causative agent of chancroid. In the broth medium, the growth phase of the organism can be monitored and the organisms achieve a cell density of > 10(8) CFU/ml.
Outbreak of Brucella melitensis among microbiology laboratory workers
Authors: Martin-Mazuelos E , Nogales M C , Florez C , Gómez-Mateos J M , Lozano F , Sanchez A
Published: January 2020

We report on an outbreak of laboratory-acquired brucellosis involving four technicians working at a microbiology laboratory. All cases occurred in a period of 4 months. Blood cultures and the Rose Bengal test were positive for Brucella spp. in all cases. Microagglutination was positive for Brucella spp. at titers of between 1/40 and 1/160. All patients were cured after treatment.