bluuebunny/crossref_metadata_2025 huggingface dataset:
New phage typing scheme for Vibrio cholerae O1 biotype El Tor strains
Authors: Chattopadhyay D J , Sarkar B L , Ansari M Q , Chakrabarti B K , Roy M K , Ghosh A N , Pal S C
Published: January 2020

The conventional phage typing scheme proposed by S. Basu and S. Mukerjee (Experientia 24:299-300, 1968) has been used routinely for identification of the strains at the Vibrio Phage Reference Laboratory since 1968. However, because of limitations of this scheme, a new phage typing scheme using five newly isolated phages was incorporated into the conventional scheme. A different definition of routine test dilution (almost confluent lysis) was found to be more useful than the one previously used (confluent lysis). The 1,000 strains tested could be clustered into 27 types with the five new phages. With the new scheme of 10 phages (5 new phages and 5 phages of Basu and Mukerjee), the 1,000 strains could be grouped into 146 types. The new phages were different from each other and also from those of Basu and Mukerjee, as revealed by lytic pattern, electron microscopy, restriction endonuclease digestion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antiphage antiserum studies. With the new typing scheme, 99.6% of the strains were typeable. Phage type 115 was the most common and includes 119 (11.9%) of the 1,000 strains tested. Next most common were phage types 142 (9.4%), 143 (7.0%), 104 and 116 (both 5.4%), 3 (5.3%), 5 (4.1%), 4 (3.9%), 24 (2.1%), and 100 (1.7%). The larger number of types would be useful for further classification of the strains for epidemiological purposes. This newly developed scheme is highly applicable to, and could be widely adopted for, phage typing of Vibrio cholerae O1 biotype El Tor strains.
Rapid methods for identification of Staphylococcus aureus when both human and animal staphylococci are tested: comparison with a new immunoenzymatic assay
Authors: Guardati M C , Guzmàn C A , Piatti G , Pruzzo C
Published: January 2020

A new immunoenzymatic assay (IEA) for the identification of Staphylococcus aureus strains of both human and animal origin was compared with rapid commercial kits. The sensitivities and specificities of the commercial kits varied from 90.2 to 96% and 90.8 to 93.7%, respectively. The IEA did not give any false-negative or false-positive results, while commercial kits gave high percentages of false-positive results among clumping factor-positive non-S. aureus strains. The IEA is particularly useful for isolates for which identification is doubtful, for large-scale epidemiological studies, and for identifying isolates from animals as S. aureus.
Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization
Authors: Yuen L K , Ross B C , Jackson K M , Dwyer B
Published: January 2020

A total of 41 Mycobacterium tuberculosis strains from patients of Vietnamese origin were analyzed by Southern blot hybridization with two different probes, IS6110 (Otal, I., et al., J. Clin. Microbiol. 29:1252-1254, 1991; Ross, B. C., et al., J. Clin. Microbiol. 30:942-946, 1992; Thierry, D., et al., J. Clin. Microbiol. 28:2668-2673, 1990; van Soolingen, D., et al., J. Clin. Microbiol. 29:2578-2586, 1991) and pTBN12 (Ross, B. C., et al., J. Clin. Microbiol. 30:942-946, 1992). The restriction fragment patterns of nine of these strains were virtually identical when the pTBN12 probe was used; five strains had a single copy of IS6110, and four strains failed to hybridize with the IS6110 probe. This relatively high frequency of strains with no or one copy of IS6110 suggests that the usefulness of IS6110 for epidemiological study may be limited in certain populations.
Screening for cephalosporin-resistant Streptococcus pneumoniae with the Kirby-Bauer disk susceptibility test
Authors: Friedland I R , Shelton S , McCracken G H
Published: January 2020

Kirby-Bauer disk susceptibility tests with five standard cephalosporin disks were performed on 23 penicillin-resistant Streptococcus pneumoniae isolates for which ceftriaxone MICs were 0.125 to 4 micrograms/ml. Cefuroxime disk inhibition zone diameters distinguished clearly isolates for which ceftriaxone MICs were > or = 2 micrograms/ml from more susceptible strains, whereas cephalothin, ceftizoxime, cefotaxime, and ceftriaxone disks distinguished these isolates less clearly than the cefuroxime disk did.
Neutralizing antibodies in cats infected with feline immunodeficiency virus
Authors: Tozzini F , Matteucci D , Bandecchi P , Baldinotti F , Siebelink K , Osterhaus A , Bendinelli M
Published: January 2020

Sera from cats experimentally infected with five isolates of feline immunodeficiency virus (FIV) from various geographical regions and from FIV enzyme-linked immunosorbent assay-seropositive field cats from four European countries neutralized the Petaluma strain of FIV (FIV-P), originally isolated in California, at high titers. In addition, FIV-P and a European isolate proved equally susceptible to neutralization by all sera tested. Coupled with observations by Fevereiro et al. (M. Fevereiro, C. Roneker, A. Laufs, L. Tavares, and F. de Noronha, J. Gen. Virol. 72:617-622, 1991), these findings indicate that most if not all FIV strains circulating in Europe and the United States share important neutralization-inducing epitopes.
Cytomegalovirus excretion in pregnant and nonpregnant women
Authors: Shen C Y , Chang S F , Yen M S , Ng H T , Huang E S , Wu C W
Published: January 2020

Cervical and urinary excretion of cytomegalovirus by Taiwanese women was identified by the presence of a cytomegalovirus-specific immediate-early gene sequence amplified by the polymerase chain reaction. Excretion rates during the first trimester of pregnancy resembled rates for nonpregnant women. As pregnancy proceeded, the cervical excretion rate increased from 13 to 40% and the urinary excretion rate increased from 1 to 13%.
Proliferation of rat Pneumocystis carinii on cells sheeted on microcarrier beads in spinner flasks
Authors: Lee C H , Bauer N L , Shaw M M , Durkin M M , Bartlett M S , Queener S F , Smith J W
Published: January 2020

A method of growing rat Pneumocystis carinii with human embryonic lung fibroblasts (HEL-299 cells) sheeted onto microcarrier beads has been developed. This method allows production of large quantities of P. carinii organisms with very little contamination of host cells. A fivefold increase in the numbers of organisms was achieved, as determined by organism count, antigen detection, and DNA quantification. The majority of organisms produced by this method are trophozoites.
Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction
Authors: Forbes B A , Hicks K E
Published: January 2020

The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 microliters) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 microM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.
Ability of clinical laboratories to detect antimicrobial agent-resistant enterococci
Authors: Tenover F C , Tokars J , Swenson J , Paul S , Spitalny K , Jarvis W
Published: January 2020

To test the ability of clinical laboratories to detect antimicrobial resistance among enterococci, we sent four vancomycin-resistant enterococcal strains and one beta-lactamase-producing enterococcus to all 93 nongovernment, hospital-based clinical laboratories in New Jersey; 76 (82%) participated in the study. Each organism was tested by the laboratory's routine antimicrobial susceptibility testing method. The proportion of laboratories that correctly reported that an isolate was resistant to vancomycin varied according to the resistance level of the isolate: high-level resistance (MIC for Enterococcus faecium = 512 micrograms/ml), 96% of laboratories correct; moderate-level resistance (MIC for E. faecium = 64 micrograms/ml), 27% correct; low-level resistance (MIC for Enterococcus faecalis = 32 micrograms/ml), 16% correct; and intrinsic low-level resistance (MIC for Enterococcus gallinarum = 8 micrograms/ml), 74% correct. The beta-lactamase-producing E. faecalis isolate was identified as resistant to penicillin and ampicillin by 66 and 8% of laboratories, respectively, but only three laboratories recognized that it was a beta-lactamase producer. This survey suggests that many laboratories may fail to detect antimicrobial agent-resistant enterococci.
Virulence-associated plasmids in Rhodococcus equi
Authors: Takai S , Watanabe Y , Ikeda T , Ozawa T , Matsukura S , Tamada Y , Tsubaki S , Sekizaki T
Published: January 2020

Twenty-three strains of Rhodococcus equi from independent clinical cases were analyzed for the presence of virulence plasmid DNA. Of the clinical isolates, 19 contained an 85-kb plasmid and the remaining 4 contained a 90-kb plasmid. All of the isolates expressed 15- to 17-kDa antigens and were virulent in mice. Restriction enzyme and Southern blot analyses showed large regions of DNA homology between the 85- and 90-kb virulence plasmids. It was concluded tentatively that there are at least two virulence plasmids in R. equi and that they have a common origin.
Characterization of two DNA probes specific for Serpulina hyodysenteriae
Authors: Sotiropoulos C , Smith S C , Coloe P J
Published: January 2020

Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology.
Diagnosis of Helicobacter pylori infection in adults and children by using the Malakit Helicobacter pylori, a commercially available enzyme-linked immunosorbent assay
Authors: Blecker U , Lanciers S , Hauser B , Vandenplas Y
Published: January 2020

Malakit Helicobacter pylori (Biolab, Limal, Belgium) is a second-generation enzyme-linked immunosorbent assay (ELISA) for the detection of Helicobacter pylori infection. We evaluated its ability to diagnose H. pylori infection in 489 asymptomatic pregnant women, 427 asymptomatic children, and 95 symptomatic children. 87 asymptomatic adults (17.8%), 31 asymptomatic children (7.3%), and 27 symptomatic children (28.4%) were seropositive. We observed an increase in H. pylori infection with age. 13C-urea breath tests were performed for all seropositive and 100 randomly selected seronegative asymptomatic adults. They were also performed for all seropositive and 65 randomly chosen seronegative asymptomatic children. Breath tests were positive for 86 of 87 (98.9%) seropositive adults, 30 of 31 seropositive children (96.8%), and no seronegative individual. Compared with those of culture, the sensitivity and specificity of the Malakit Helicobacter pylori were both 96%. We conclude that the Malakit Helicobacter pylori is equally suitable for adults and children. Therefore, this ELISA can be proposed as an important alternative to other more time-consuming and/or more expensive diagnostic tests for the detection of H. pylori.
Herpes simplex virus detection from genital lesions: a comparative study using antigen detection (HerpChek) and culture
Authors: Cone R W , Swenson P D , Hobson A C , Remington M , Corey L
Published: January 2020

The sensitivity of a rapid enzyme immunoassay test (HerpChek Direct Herpes Simplex Virus Antigen Test [DuPont Medical Products, Wilmington, Del.]) for the detection of herpes simplex virus (HSV) antigens in patient specimens was compared with HSV culture. HerpChek positivity for HSV occurred with 179 (65%) of 275 lesion specimens, whereas culture for HSV was positive for 145 (53%) lesions (P = 0.002). HerpChek was twice as sensitive as culture for the detection of HSV in late-stage lesions and was equivalent to culture for the detection of HSV in early lesions. We conclude that HerpChek provides greater sensitivity than culture for HSV detection in late-stage genital lesions.
A phaeohyphomycotic cyst and peritonitis caused by Phialemonium species and a reevaluation of its taxonomy
Authors: King D , Pasarell L , Dixon D M , McGinnis M R , Merz W G
Published: January 2020

Two cases of human fungal infections caused by members of the genus Phialemonium, a genus proposed by Gams and McGinnis (1983) for fungi intermediate between the genera Acremonium and Phialophora, are presented. The first case was a phaeohyphomycotic cyst on the foot of a renal transplant recipient. The fungus was detected by direct examination and histopathology and was recovered by several procedures over 4 months. It was flat, glabrous, and white becoming yellow with the production of a diffusible yellow pigment; it had conidiophores that were mostly solitary and lateral and terminal phialides and adelophialides with distinct collarettes producing cylindrical to curved conidia. The isolate resembled both Phialemonium dimorphosporum and Phialemonium curvatum, although its characteristics were more consistent with those of the latter. The second case was peritonitis in a renal transplant recipient. The fungus was white-to-cream colored and yeast like, but later became black with a green diffusible pigment, and produced obovoid conidia; it was easily identified as Phialemonium obovatum. Difficulties encountered in the identification and taxonomy of members of this genus highlight the need for standardized conditions, e.g., potato dextrose agar culture incubated at 24 to 25 degrees C for morphologic comparisons, to control significant variations due to culture conditions.
Identification of Actinobacillus actinomycetemcomitans by leukotoxin gene-specific hybridization and polymerase chain reaction assays
Authors: Tønjum T , Haas R
Published: January 2020

Eleven strains of Actinobacillus actinomycetemcomitans isolated from cases of systemic infections, local abscesses, and periodontitis were identified by genetic assays using the leukotoxin gene as the target. We have developed a polymerase chain reaction (PCR) assay, based on the leukotoxin structural gene of this pathogen, which clearly identified all tested strains of A. actinomycetemcomitans and separated them from the closely related Haemophilus aphrophilus as well as other bacterial species. Furthermore, DNA-DNA hybridization was performed with the cloned partial leukotoxin structural gene (lktA) as a probe, which again clearly distinguished A. actinomycetemcomitans from H. aphrophilus, parts of the normal oral flora, and species harboring RTX (repeats in toxin) family-related cytotoxins. The PCR fragment amplified from the leukotoxin structural gene gave results similar to those given by the cloned leukotoxin gene when used as a probe in hybridization experiments. The hybridization and PCR assays described here are fundamental improvements for the identification of A. actinomycetemcomitans.
Development and evaluation of a blood-free medium for determining growth curves and optimizing growth of Rochalimaea henselae
Authors: Schwartzman W A , Nesbit C A , Baron E J
Published: January 2020

Two strains of Rochalimaea henselae were used to optimize a blood-free growth medium. Seven agar bases, four broths, and combinations of eight supplements were evaluated. Acceptable growth was achieved in media containing Fildes solution and hemin, with the best growth demonstrated in brucella broth or on brucella agar with 6 to 8% Fildes solution and 250 micrograms of hemin per ml. R. henselae utilized hemin in concentrations six times that utilized by Rochalimaea quintana. Erythrocyte membrane was necessary to achieve the full growth-promoting effect of rabbit blood.
Total cellular fatty acid composition of cultured Pneumocystis carinii
Authors: Paulsrud J R , Queener S F , Bartlett M S , Smith J W
Published: January 2020

Oleic acid makes up > 50% of the total fatty acids of Pneumocystis carinii grown on WI-38 cells. Oleic acid levels increased in parallel with increasing trophozoites over 7 days in culture. The fatty acid composition of P. carinii resembles that of certain fungi but differs from those of lung surfactant lipid, host cells, and fetal bovine serum.
Inhibition of Acanthamoeba species by Pseudomonas aeruginosa: rationale for their selective exclusion in corneal ulcers and contact lens care systems
Authors: Qureshi M N , Perez A A , Madayag R M , Bottone E J
Published: January 2020

Cocultivation of Acanthamoeba castellanii and Acanthamoeba polyphaga with live Pseudomonas aeruginosa and with broth filtrates of P. aeruginosa proved equally lethal to the Acanthamoeba spp. The P. aeruginosa-induced amebicidal activity is apparently toxin mediated and has two operative modes: it can function through binding of P. aeruginosa to the ameba membrane and in the presence of one or more P. aeruginosa exoproducts.
Catheter-related Rahnella aquatilis bacteremia in a pediatric bone marrow transplant recipient
Authors: Hoppe J E , Herter M , Aleksic S , Klingebiel T , Niethammer D
Published: January 2020

Rahnella aquatilis, a rarely encountered member of the family Enterobacteriaceae, was twice isolated from the blood of a pediatric bone marrow transplant recipient. This is the first report of a pediatric case of R. aquatilis bacteremia, and it was probably related to inappropriate handling of a Hickman catheter.
Evaluation of the L-pyrrolidonyl-beta-naphthylamide hydrolysis test for the differentiation of members of the families Enterobacteriaceae and Vibrionaceae
Authors: Chagla A H , Borczyk A A , Aldom J E , Dalla Rosa S , Cole D D
Published: January 2020

A simple, rapid, and inexpensive spot test incorporating the substrate pyrrolidonyl naphthylamide was used to examine pyrrolidonyl peptidase activity among 800 bacterial strains belonging to the families Enterobacteriaceae and Vibrionaceae. The pyrrolidonyl naphthylamide test was found to be particularly useful in separating Citrobacter spp. (100% positive) from Salmonella spp. (0.4% positive) and Escherichia coli (0% positive). Furthermore, it would appear to offer a safer alternative to the traditional potassium cyanide test for differentiating citrobacters from salmonellae.
N-acetyl-D-glucosamine medium improves recovery of Haemophilus influenzae from sputa of patients with cystic fibrosis
Authors: Möller L V , van Alphen L , Grasselier H , Dankert J
Published: January 2020

A modified selective medium supplemented with N-acetyl-D-glucosamine (NAG), hemin, and NAD plus two cefsulodin disks, for primary isolation of nonencapsulated Haemophilus influenzae from sputum of patients with cystic fibrosis, is described. Isolation of H. influenzae from this medium, designated NAG medium, was compared with recovery by standard media and immunochemical detection of H. influenzae with monoclonal antibody 8BD9. The H. influenzae recovery rate increased from 31% with standard media to 42% with NAG medium. H. influenzae was detected by immunoperoxidase staining in 54% of the sputum specimens. The results of this study demonstrate that NAG medium improves H. influenzae recovery, although immunoperoxidase staining is superior for detection of H. influenzae from sputum of cystic fibrosis patients.
Relationship between indole production and differentiation of Klebsiella species: indole-positive and -negative isolates of Klebsiella determined to be clonal
Authors: Maslow J N , Brecher S M , Adams K S , Durbin A , Loring S , Arbeit R D
Published: January 2020

Klebsiellae are an important cause of nosocomial infections. The two clinically relevant species, Klebsiella pneumoniae and Klebsiella oxytoca, are differentiated by the ability to produce indole from tryptophan, K. oxytoca being indole positive. We report here the detailed biochemical and molecular analysis of two isolates of Klebsiella, cultured from the same urine specimen, that differed only in their ability to produce indole. The two isolates were identical as determined by ribotyping and pulsed-field gel electrophoresis, and they differed from 10 epidemiologically unrelated strains. Probing with the Escherichia coli tryptophanase operon, tna, revealed seven restriction fragment length polymorphisms (RFLP) among the 12 strains. The two index strains had identical RFLP; no single RFLP could account for all of the indole-positive or -negative strains. Thus, the identification of epidemiologically related strains of Klebsiella differing only in indole production may warrant further examination to determine whether the strains are clonal.
Characterization of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type-specific cDNA probes
Authors: Parwani A V , Hussein H A , Rosen B I , Lucchelli A , Navarro L , Saif L J
Published: January 2020

Dot and Northern blot hybridization assays were used to analyze field strains of group A bovine rotaviruses (BRVs) by using nucleic acid probes representing P and G type specificities. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions of the cloned VP4 (nucleotides 211 to 686) and VP7 (nucleotides 51 to 392) genes from four serotypically distinct (in P or G types) strains of rotaviruses: NCDV (G6, P1), IND (G6, P5), 69M (G8, P10), and Cr (G10, P11). The P and G type cDNA probes were radiolabeled with [32P]dCTP and hybridized with RNA extracted from reference cell culture-passaged rotavirus strains or the field samples. The field samples were obtained from young diarrheic calves from Ohio, Nebraska, Washington State, and Canada. The cDNA probes were specific for their respective G or P types on the basis of analysis of known P and G type reference strains. The G typing analysis of 102 field samples revealed that 36.3% (37 of 102) were G6, 2.9% (3 of 102) were G8, 12.7% (13 of 102) were G10, and 23.5% (24 of 102) were untypeable. The P typing results for 93 samples indicated that 2.2% (2 of 93) were P1 (NCDV-like), 20.4% (19 of 93) were P5 (UK-like), 9.3% (10 of 93) were P11 (B223-like), and 40.8% (38 of 93) were untypeable. This is the first report of the identification among BRV strains in North America of a G type other than G6 or G10. Our report further confirms that G6, P5 rotaviruses are predominant among the BRV field strains that we examined, and the P types of these strains differ from that of the BRV vaccine strain used in the United States (G6, P1). The large number of untypeable G (23.5%) and P (40.8%) types suggests that other or new P and G types exist among BRV field strains.
Rapid detection of vesicular stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction
Authors: Rodriguez L L , Letchworth G J , Spiropoulou C F , Nichol S T
Published: January 2020

Vesicular stomatitis virus of the New Jersey serotype (VSV-NJ) causes vesicular disease in cattle, pigs, and horses throughout the Americas. Vesicular disease is clinically indistinguishable from foot-and-mouth disease (FMD). Therefore, outbreaks of vesicular disease in FMD-free areas must be rapidly diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm the absence of FMD. Diagnosis is currently performed in high-containment (biosafety level 3) laboratories by using complement fixation and virus isolation in tissue culture. We describe here an alternative method for the detection of VSV-NJ RNA in clinical samples. This method includes a rapid acid guanidine-phenol RNA extraction procedure coupled with a one-tube polymerase chain reaction (PCR) using reverse transcriptase. By using this test, we were able to detect the largest number of positive samples (53 of 58), followed by complement (48 of 58) and isolation in tissue culture (43 of 58). The primers chosen for this assay amplify a 642-nucleotide region of the phosphoprotein gene of VSV-NJ but not of VSV-IN. Sequencing of the PCR product enables genetic typing of virus isolates and epidemiological studies. Since no infectious materials are necessary to perform this test and any infectious virus in clinical samples is destroyed by acid guanidine-phenol treatment, diagnosis can be safely performed in regular diagnostic laboratories.
Isolation and characterization of Borrelia burgdorferi from blood of a bird captured in the Saint Croix River Valley
Authors: McLean R G , Ubico S R , Hughes C A , Engstrom S M , Johnson R C
Published: January 2020

Field investigations were conducted to further evaluate the role of birds in the maintenance and dissemination of Borrelia burgdorferi. Blood specimens were taken from 39 passerine birds of 17 species captured during June 1991 at the Saint Croix National Riverway in Wisconsin, and one isolate, WI91-23, was cultured from an adult song sparrow (Melospiza melodia). This isolate was shown to be infectious for Peromyscus leucopus and Mesocricetus auratus (golden hamster). Isolate WI91-23 was confirmed as B. burgdorferi by immunofluorescence assay by using species-specific anti-OspA monoclonal antibodies H3TS and H5332 and anti-OspB antibody H5TS. Isolate WI91-23 was compared with Borrelia anserina Es, Borrelia hermsii MAN-1, and other B. burgdorferi strains (ATCC 53210, CT-1, and Catharus fuscescens [veery] liver 10293). Pulsed-field gel electrophoresis of in situ-lysed spirochetes revealed that the DNA plasmid profile of WI91-23 was most similar to those of plasmids from B. burgdorferi and most different from those of plasmids from B. anserina and B. hermsii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the protein profile of WI91-23 was like that of other B. burgdorferi strains studied, with dominant proteins corresponding to OspA and OspB, and that it differed from the protein profiles of B. anserina and B. hermsii. These findings indicate that passerine birds may serve as reservoirs for B. burgdorferi.
Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts
Authors: Newman R D , Jaeger K L , Wuhib T , Lima A A , Guerrant R L , Sears C L
Published: January 2020

The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) (LMD Laboratories, Carlsbad, Calif.) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate-concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection.
Comparative chemotaxonomic studies of mycolic acid-free coryneform bacteria of human origin
Authors: Barreau C , Bimet F , Kiredjian M , Rouillon N , Bizet C
Published: January 2020

Forty-two clinical isolates were classified as Corynebacterium minutissimum, Corynebacterium striatum, and Corynebacterium CDC group I by the API Coryne system. The chemotaxonomic characteristics of the isolates were determined by thin-layer chromatographic analysis. Twenty-six isolates were found to have a type IV cell wall (meso-di-aminopimelic acid arabinose, galactose) but did not contain mycolic acids. These 26 isolates shared chemotaxonomic characteristics with those of mycolic acid-free reference strains (including the Corynebacterium amycolatum NCFB 2768 type strain, "Corynebacterium asperum," and coryneform CDC groups I2 and F2). The total protein profiles of the isolates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were similar to each other and to that of the C. amycolatum type strain. The profiles of the reference strains "Corynebacterium asperum" (CIP 100836, CIP 80.54, CIP 79.37, CIP 52.13), coryneform bacteria CDC groups I2 and F2 (CDC F5771, F5890, G723, G1970), and C. amycolatum were closely related. Thus, the mycolic acid-negative strains with a chemotype IV wall may belong to a single taxon. DNA hybridization studies could confirm this hypothesis. The present study shows the importance of chemotaxonomic analysis for verifying strain identifications and completing results from biochemical tests, particularly for coryneform bacteria.
Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission
Authors: Eriks I S , Stiller D , Palmer G H
Published: January 2020

Anaplasma marginale, an intraerythrocytic rickettsia of cattle, is transmitted biologically by ticks. Because of the brevity of acute A. marginale infection, transmission may rely on the tick's ability to acquire the organism from persistently infected cattle with low rickettsemia levels. By using a nucleic acid probe to quantitate low-level infection, we found that rickettsemia levels in persistently infected cattle fluctuated at approximately 5-week intervals during a 24-week period, from < 10(4) infected erythrocytes per ml of blood to high levels of approximately 10(7) infected erythrocytes per ml of blood. Cattle maintained very low rickettsemia levels (< 10(4.3) infected erythrocytes per ml of blood) for approximately 4 to 8 days of every 5-week cycle. The effect of fluctuations in rickettsemia in persistently infected cattle on acquisition by Dermacentor andersoni nymphal and adult male ticks was examined. A positive correlation was observed between rickettsemia levels in cattle and the resulting infection rates of ticks. At high rickettsemia levels, up to 80% of ticks acquired infection, but even at extremely low rickettsemia levels, 27% of adult male ticks became infected. Moreover, once ticks acquired infection, biological replication of the organism within the ticks appeared to make up for initial differences in the infecting dose. The high infection rates in adult males, combined with their intermittent feeding behavior and the observation that only a few infected ticks were required for transmission to a susceptible host, suggest that adult male D. andersoni ticks are epidemiologically important in A. marginale transmission. Because cattle with all levels of rickettsemia were capable of efficient transmission to ticks, population control efforts must include decreasing transmission from persistently infected individuals.
Evaluation of alkaline phosphatase-labelled ipaH probe for diagnosis of Shigella infections
Authors: Oberhelman R A , Kopecko D J , Venkatesan M M , Salazar-Lindo E , Gotuzzo E , Yi A , Chea-Woo E , Ruiz R , Fernandez-Prada C , León-Barúa R
Published: January 2020

The presence of many enteropathogens which are not easily detectable by routine stool culture has led to the development of alternative diagnostic methods. One of these techniques, nucleic acid probe hybridization, has been used to identify Shigella spp. and enteroinvasive Escherichia coli (EIEC) in stool specimens through the detection of genetic material encoded by a specific large approximately 200-kbp virulence-related plasmid. In the present study, an alkaline phosphatase-labelled oligonucleotide probe developed to detect the gene for ipaH, a repetitive genetic sequence thought to be present on both the virulence-related plasmid and the chromosomes of all strains of Shigella and EIEC, was tested in a developing-country setting through a prospective clinical trial. In a group of 219 Peruvian adults and children with acute gastroenteritis, the ipaH probe detected 85% of cases of shigellosis and demonstrated a specificity of 95% when compared with simultaneous detection by several stool culture techniques. Additionally, three cases of EIEC infection which could not be diagnosed by culture methods alone were detected with the ipaH probe and were confirmed by plasmid analysis and Sereny testing. These preliminary results suggest that, with further research, the ipaH probe should prove to be a useful and rapid adjunct in the diagnosis of acute gastroenteritis in developing countries.
Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2, and D)
Authors: Luk J M , Kongmuang U , Reeves P R , Lindberg A A
Published: January 2020

Many parts of the Salmonella rfb gene clusters which are responsible for biosynthesis of the oligosaccharide-repeating units of the O-antigenic lipopolysaccharide have recently been cloned and sequenced. On the basis of this knowledge, three sets of nucleotide primers were selected to target defined regions of the abequose and paratose synthase genes: rfbJ of Salmonella serogroup B, rfbJ of Salmonella serogroup C2, and rfbS of Salmonella serogroup D (also present in serogroup A). For good differentiation among these major serogroups, the primers were designed not only to give precise specificity in priming but also to give DNA products with different sizes in polymerase chain reactions (product sizes, approximately 720 bp for both serogroups A and D, approximately 820 bp for serogroup C2, and approximately 882 bp for serogroup B). In a polymerase chain reaction assay utilizing these rfb-specific primers, all of the 40 salmonellae belonging to serogroups B, C2, and D plus A were accurately identified among a total of 123 clinical isolates tested (including 55 salmonellae from 36 different serotypes and 68 strains from 10 other members of the family Enterobacteriaceae). No false-positive reactions were detected. The selected rfb gene sequences were proved for the first time to be useful DNA-based markers for identification of and differentiation among Salmonella serogroups A, B, C2, and D.
Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis
Authors: Goldbaum F A , Leoni J , Wallach J C , Fossati C A
Published: January 2020

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.
Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations
Authors: Green K Y , Lew J F , Jiang X , Kapikian A Z , Estes M K
Published: January 2020

Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness.
Biochemical heterogeneity of Mycobacterium tuberculosis complex isolates in Guinea-Bissau
Authors: Hoffner S E , Svenson S B , Norberg R , Dias F , Ghebremichael S , Källenius G
Published: January 2020

Fifty-six strains of the Mycobacterium tuberculosis complex from patients in Guinea-Bissau were examined by using four biochemical tests (niacin production, nitrate reductase, pyrazinamidase, and resistance to thiophen-2-carboxylic acid hydrazide). The isolates were divided into five different biovars within a spectrum ranging from classical human M. tuberculosis to classical M. bovis.
Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital
Authors: Claas E C , van Milaan A J , Sprenger M J , Ruiten-Stuiver M , Arron G I , Rothbarth P H , Masurel N
Published: January 2020

A prospective clinical evaluation of the reverse transcriptase polymerase chain reaction (RNA PCR) for detection of influenza viruses was carried out with specimens from 342 patients of a children's hospital in The Netherlands. The RNA PCR, carried out directly on the specimens without an organic extraction, showed a sensitivity and specificity which are superior to those of direct immunofluorescence and comparable to those of cell culture combined with immunofluorescence (culture/IF). Negative results can be obtained within 2 days by the RNA PCR but may take up to 14 days by culture/IF. Because culturing is the standard technique for the detection of respiratory viruses, at this moment there are no strong arguments to replace culture/IF with RNA PCR for the detection of influenza A virus.
Application and staining patterns of commercial anti-Pneumocystis carinii monoclonal antibodies
Authors: Elvin K , Linder E
Published: January 2020

Commercially available monoclonal antibodies to Pneumocystis carinii were compared with respect to immunofluorescence staining patterns of human immunodeficiency virus-inactivated smears. Only the indirect staining kits were suitable for application to ethanol-inactivated samples. When antibodies from Dakopatts and Northumbria were compared, the staining of cysts and trophozoites showed different patterns.
Application of rejection criteria for stool cultures for bacterial enteric pathogens
Authors: Fan K , Morris A J , Reller L B
Published: January 2020

Over 20 months, we prospectively assessed the yield of cultures for Salmonella, Shigella, and Campylobacter spp. from adults and children. In the first 10 months, 53% (567 of 1,097) of specimens from adults were from patients who had been in the hospital for > 3 days. Overall, only 2.6% (29 of 1,097) of specimens contained pathogens, and all were from patients who had been in the hospital for < or = 3 days. Over the second 10 months, specimens from adults in the hospital for > 3 days were not cultured unless special reasons existed. Thirty percent (368 of 1,229) of specimens were rejected. Concurrently, 7.5% (51 of 677) of stool specimens from children were positive. Only one positive specimen came from a child who had been in the hospital for > 3 days. Neither stool consistency nor fecal leukocytes were useful predictors for the presence of an enteric pathogen. We recommend that specimens from both adults and children in the hospital for > 3 days not be cultured unless there are delineated plausible clinical or epidemiological reasons to do so.
Seroepidemiological study of African horse sickness virus in The Gambia
Authors: Staeuber N , Fye B , Zinsstag J , McCullough K C
Published: January 2020

An indirect enzyme-linked immunosorbent assay was used for the screening of horse sera from The Gambia for antibodies against African horse sickness virus (AHSV). The AHSV antigen used for coating was semipurified according to the method of Manning and Chen (Curr. Microbiol. 4:381, 1980); control mock-infected Vero cell antigen was treated in the same manner. A total of 459 horse serum samples were assayed at a single dilution (1:10), and their reactivities were compared with those of reference positive anti-AHSV and reference negative horse sera. A total of 81% of the horse serum samples clearly contained antibodies against AHSV; this consisted of 18% (of the total number of serum samples) strongly positive, 46.5% moderately positive, and 16.5% weakly but still clearly positive. Such results suggest a high prevalence of AHSV in the regions from whence the samples originated. Reports from investigations in other countries in this area of West Africa have also shown a high prevalence for anti-AHSV antibodies in equids. The question is raised as to how the animals became seropositive and whether the observations represent an increased resistance of horses living in a region in which AHS is enzootic.
Comparison of rapid diagnostic techniques for respiratory syncytial and influenza A virus respiratory infections in young children
Authors: Dominguez E A , Taber L H , Couch R B
Published: January 2020

We performed virus isolation tests for respiratory viruses on combined nasal wash-throat swab specimens collected from infants and children with acute respiratory illnesses presenting to a hospital clinic during a 3-month period of concurrent epidemics of respiratory syncytial virus (RSV) and influenza A virus (Flu A) infections. Virus isolation results were used to assess the utility of commercially available rapid diagnostic kits for these two viruses. The kits employed direct immunofluorescence (IF) of cells (Imagen for RSV and Flu A), indirect IF of cells (Baxter Bartels Microscan), and enzyme immunoassay (EIA) (Becton Dickinson Directigen for RSV and Flu A and Abbott TestPack for RSV). All testing was completed on 81 specimens from 80 subjects. Of the 81 specimens, 53 (65%) yielded a virus: RSV, 28%; Flu A, 25%; rhinovirus, 6%; and enterovirus, cytomegalovirus, herpes simplex virus, and adenovirus, 2 to 4% each. Among the tests, Bartels Microscan and Directigen Flu-A exhibited the highest sensitivities (87 and 75%) and efficiencies (94 and 94%) for RSV and Flu A, respectively. All the tests exhibited high specificity. Thus, optimal detection of RSV and Flu A among infants and children who presented to a hospital clinic required two different detection methods (IF and enzyme immunoassay) and kits from two different companies (Baxter [Bartels Microscan] and Becton Dickinson [Directigen]).
Combining pooling and alternative algorithms in seroprevalence studies
Authors: Raboud J M , Sherlock C , Schechter M T , Lepine D G , O'Shaughnessy M V
Published: January 2020

Data from two seroprevalence studies and one comparative study of confirmatory algorithms were used to compare the costs and sensitivities of six algorithms for determining seropositivity to human immunodeficiency virus (HIV). We evaluated confirmatory strategies by using the CBC Recombigen HIV enzyme immunoassay (EIA; Cambridge BioScience, Worcester, Mass.) and immunoblotting followed by radioimmunoprecipitation assay to confirm indeterminate immunoblotting results with and without pooling of samples during screening. The least expensive algorithm was that in which sera were pooled during screening and EIA was used to confirm positive test results. The cost savings associated with this confirmatory test were greater when the prevalence of HIV infection was higher. Savings from pooling of sera for screen testing diminished as HIV prevalence increased. The sensitivity and specificity of EIA with respect to immunoblotting and radioimmunoprecipitation assay were estimated to be 0.9992 and 0.9977, respectively. We found that the implementation of pooling during screening and the use of EIA as the confirmatory test do not affect the statistical reliability of estimates of seropositivity but do result in considerable cost savings.
Restriction endonuclease analysis and ribotyping differentiate Pasteurella haemolytica serotype A1 isolates from cattle within a feedlot
Authors: Murphy G L , Robinson L C , Burrows G E
Published: January 2020

Pasteurella haemolytica serotype A1 isolates were collected from cattle within a feedlot during an outbreak of bovine respiratory disease. Genetic heterogeneity among the isolates was examined by restriction endonuclease analysis (REA), ribotyping, and analysis of plasmid content. The susceptibilities of isolates to several antibiotics were also examined. Five different REA patterns and three different ribotypes were observed among the isolates. Fifty percent of the isolates had an identical REA type, ribotype, and plasmid profile. Examination of the plasmid content of the isolates revealed that most (73%) carry a single plasmid which encodes beta-lactamase, 13.5% carry two plasmids, and 13.5% carry no plasmid. The data reveal the presence of genetic differences among isolates of P. haemolytica A1, associated with shipping fever pneumonia within a closed feedlot, and suggest that a combination of REA, ribotyping, plasmid analysis, and antibiotic susceptibility determination will be useful in analyzing the molecular epidemiology of this disease.