bluuebunny/crossref_metadata_2025 huggingface dataset:
A 4-year survey of antimicrobial susceptibility trends for isolates from cattle with bovine respiratory disease in North America
Authors: Watts J L , Yancey R J , Salmon S A , Case C A
Published: January 2020

The antimicrobial susceptibility trends of bovine respiratory disease (BRD) pathogens isolated from 1988 to 1992 were determined. A total of 880 isolates representing Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus were used in the study. Overall, resistance to ampicillin, tetracycline, erythromycin, and sulfamethazine was frequently encountered among strains of P. haemolytica and P. multocida. Ceftiofur, an extended-spectrum cephalosporin originally marketed in 1988 for the treatment of BRD, was very active against the BRD pathogens tested; the MIC of ceftiofur for 90% of isolates tested was < or = 0.06 microgram/ml. Resistance to spectinomycin varied on the basis of the breakpoint used. Substantial variation in the year-to-year susceptibility of BRD pathogens to tilmicosin, a new macrolide antimicrobial agent, was observed. The proportion of susceptible P. haemolytica isolates ranged from 84.7% in the second year to 7.1% in the third year and 78.2% in the fourth year. Similar fluctuations were observed with strains of P. multocida.
Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral Leishmaniasis in immunocompromised patients
Authors: Piarroux R , Gambarelli F , Dumon H , Fontes M , Dunan S , Mary C , Toga B , Quilici M
Published: January 2020

A PCR assay amplifying a repeated sequence from the Leishmania infantum genome was compared with direct examination of bone marrow aspirate, myeloculture, and serology for the diagnosis of visceral leishmaniasis in immunocompromised patients. Of 73 patients living in an area endemic for leishmaniasis and where visceral leishmaniasis was suspected by physicians, only 10 had an indisputable diagnosis of visceral leishmaniasis. None of the diagnostic tests performed in the study achieved 100% sensitivity for diagnosing visceral leishmaniasis. PCR exhibited superior sensitivity (82%) in comparison with bone marrow aspirate examination (55%) and myeloculture (55%). Our PCR assay also showed good specificity (97%), negative predictive value (97%), and positive predictive value (82%) even when all unconfirmed PCR results were scored as false positives. Serology exhibited good sensitivity (80%) and excellent specificity (100%), negative predictive value (98%), and positive predictive value (100%) in diagnosing new cases of visceral leishmaniasis but failed to diagnose relapses. We also observed consistent negative serological results using several different immunological detection methods for 2 of the 10 patients with confirmed cases of visceral leishmaniasis. This lack of serological reactivity persisted throughout the course of their infections. These results demonstrate the importance of using PCR as an aid in the diagnosis of visceral leishmaniasis in immunocompromised patients.
Effect of simultaneous administration of cold-adapted and wild-type influenza A viruses on experimental wild-type influenza infection in humans
Authors: Youngner J S , Treanor J J , Betts R F , Whitaker-Dowling P
Published: January 2020

On the basis of the ability of the attenuated cold-adapted strain of influenza A virus to suppress disease production in ferrets simultaneously infected with epidemic influenza virus (P. Whitaker-Dowling, H.F. Maassab, and J.S. Youngner, J. Infect. Dis. 164:1200-1202, 1991), an evaluation of the ability of the cold-adapted virus to modify clinical disease in humans was made. Adult volunteers with prechallenge serum hemagglutination-inhibition titers to the influenza A/Kawasaki/86 (H1N1) virus of < or = 1:8 received either 10(7) 50% tissue culture infective doses of the wild-type A/Kawasaki virus or a mixture of 10(7) 50% tissue culture infective doses of each of the wild-type virus and a cold-adapted A/Kawasaki reassortant virus by intranasal drops in a randomized, double-blind fashion. Symptoms and wild-type virus shedding were assessed daily for 6 days following challenge. Results were compared with those derived from another group of volunteers who received only cold-adapted virus. Volunteers who received the mixed inoculum of cold-adapted and wild-type viruses had lower symptom scores than those who received wild-type virus alone, suggesting that coinfection with the cold-adapted virus may modify wild-type virus infection, but the differences were not statistically significant in this small study. The data demonstrate that administration of cold-adapted influenza A virus to humans at the time of wild-type virus infection is a safe procedure.
Identification of Bordetella pertussis infection by shared-primer PCR
Authors: Li Z , Jansen D L , Finn T M , Halperin S A , Kasina A , O'Connor S P , Aoyama T , Manclark C R , Brennan M J
Published: January 2020

A shared-primer PCR method for the detection of infection was developed by using primers derived from DNA sequences upstream of the structural genes for the porin proteins of Bordetella pertussis and Bordetella parapertussis. This method resulted in a 159-bp PCR product specific for B. pertussis and a 121-bp DNA fragment specific for B. parapertussis and allowed for the simultaneous detection of these pathogens. The PCR procedure was shown to be very specific since no PCR product was obtained from 36 non-Bordetella bacterial DNAs. Nasopharyngeal aspirates (NPAs) from children suspected of having pertussis were evaluated by the PCR method, culture, and the Chinese hamster ovary (CHO) cell assay, which detects pertussis toxin. B. pertussis was cultured from 119 of 205 NPAs assayed, and the presence of pertussis toxin was detected in 69 of the NPAs by the CHO cell assay. When ethidium bromide staining was used to detect PCR products, 100 NPAs gave positive results by shared-primer PCR; 94 of these NPAs were also positive by culture. The result indicated a sensitivity of 79% for PCR when culture was used as the standard. The sensitivity of PCR was increased to 95% when a digoxigenin immunoblot system was used. An additional 20 NPAs from patients with suspected pertussis that were culture negative also gave positive results by PCR. The specific and sensitive PCR method described here should be useful for both the clinical diagnosis of pertussis and case identification in vaccine trials.
Bisulfite or sulfite inhibits growth of Helicobacter pylori
Authors: Hawrylik S J , Wasilko D J , Haskell S L , Gootz T D , Lee S E
Published: January 2020

Bisulfite or sulfite was found to be inhibitory to Helicobacter pylori growth. A modified version of Brucella broth (BB), bisulfite-less BB (BLBB), supported rapid, robust, and consistent growth of H. pylori. We suggest that BLBB simply be called "Pylori broth" for distinction from Brucella broth.
Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism
Authors: Cheron M , Abachin E , Guerot E , el-Bez M , Simonet M
Published: January 2020

We demonstrate that DNA restriction fragment length polymorphism determined by pulsed-field gel electrophoresis is very useful in the investigation of the epidemiology of hospital-acquired infections caused by Alcaligenes denitrificans subsp. xylosoxydans. This approach showed that hospital-acquired infections caused by this opportunistic pathogen over a 6-month period in 10 patients hospitalized in an intensive care unit and a surgical unit were not a true outbreak. In addition, this molecular typing method established that the respiratory therapy equipment was the source of the contamination of two patients.
Animal and public health implications of gastric colonization of cats by Helicobacter-like organisms
Authors: Otto G , Hazell S H , Fox J G , Howlett C R , Murphy J C , O'Rourke J L , Lee A
Published: January 2020

The bacterial genus Helicobacter contains a number of species which colonize the gastric mucosa of mammals. Natural and/or experimental gastric pathology has been correlated with colonization in humans and a wide variety of animal species. Historical reports in the literature suggest that a high percentage of cats are colonized by large, spiral, gastric helicobacter-like organisms (GHLOs). One of these bacteria (Helicobacter felis) has been isolated on artificial media and has experimentally caused gastritis in gnotobiotic dogs. This study surveyed the prevalence of helicobacter colonization in random-source cats by using the urease assay. Histologic examination was performed to determine the degree of associated pathology present. GHLOs associated with chronic gastritis were present in 70% of the juvenile and 97% of the adult cats studied. Although further study is needed to determine specifically what role GHLOs play in feline gastrointestinal disease, these results indicate that helicobacter colonization should be considered in the pathogenesis of feline gastroenteropathy. Furthermore, the high prevalence of feline infection is interesting because cats have recently been implicated as a potential reservoir for human infection by helicobacter-like organisms.
Rapid determination of antifungal activity by flow cytometry
Authors: Green L , Petersen B , Steimel L , Haeber P , Current W
Published: January 2020

We have developed a rapid assay of antifungal activity which utilizes flow cytometry to detect accumulation of a vital dye in drug-damaged fungal cells. Results of these studies suggest that flow cytometry may provide an improved, rapid method for determining and comparing the antifungal activities of compounds with differing modes of action.
Contact hemolysin production by strains of enteroaggregative Escherichia coli isolated from children with diarrhea
Authors: Haque M A , Ohki K , Kikuchi M , Kohashi O
Published: January 2020

Forty-five strains of enteroaggregative Escherichia coli were tested for hemolytic activity with different culture media and erythrocytes from different species. Thirty-seven strains showed proteinase K-sensitive contact hemolysin activity with sheep erythrocytes when cultured in Casamino Acids-yeast extract broth supplemented with 1 mM calcium chloride and when cultured in nutrient broth media. The production of contact hemolysin was dependent on temperature, pH, and culture conditions.
Evaluation of media for determining hemolytic activity and that of API Listeria system for identifying strains of Listeria monocytogenes
Authors: Fujisawa T , Mori M
Published: January 2020

Several media were used to evaluate the hemolytic activity of Listeria monocytogenes, and this property was used along with the API Listeria system to identify Listeria spp. All L. monocytogenes strains were identified correctly with this system, and blood agar base no. 2 and Columbia blood agar base supplemented with horse blood were suitable for detection of hemolytic activity.
Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii
Authors: Herwaldt B L , Lew J F , Moe C L , Lewis D C , Humphrey C D , Monroe S S , Pon E W , Glass R I
Published: January 2020

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.
Identification of a novel rickettsial infection in a patient diagnosed with murine typhus
Authors: Schriefer M E , Sacci J B , Dumler J S , Bullen M G , Azad A F
Published: January 2020

Identification of ELB agent-infected fleas and rodents within several foci of murine typhus in the United States has prompted a retrospective investigation for this agent among human murine typhus patients. This agent is a recently described rickettsia which is indistinguishable from Rickettsia typhi with currently available serologic reagents. Molecular analysis of the 17-kDa antigen gene and the citrate synthase gene has discriminated this bacterium from other typhus group and spotted fever group rickettsiae. Current sequencing of its 16S ribosomal DNA gene indicates a homology of 98.5% with R. typhi and 99.5% with R. rickettsii. Through a combination of restriction fragment length polymorphism and Southern hybridization analysis of rickettsia-specific PCR products, one of five tested patient blood samples was shown to be infected with ELB while R. typhi infections were confirmed in the remaining samples. This is the first reported observation of a human infection by the ELB agent and underscores the utility of PCR-facilitated diagnosis and discrimination of these closely related rickettsial infections.
Serodiagnosis by passive hemagglutination test and verotoxin enzyme-linked immunosorbent assay of toxin-producing Escherichia coli infections in patients with hemolytic-uremic syndrome
Authors: Yamada S , Kai A , Kudoh Y
Published: January 2020

Eight cases of hemolytic-uremic syndrome in which no pathogens were isolated were diagnosed serologically by a passive hemagglutination assay and a verotoxin (VT; Shiga-like toxin) enzyme-linked immunosorbent assay (ELISA). The passive hemagglutination assay employed formalinized sheep erythrocytes sensitized with soluble native antigen or heat-treated antigen (lipopolysaccharide [LPS]) from Escherichia coli O26, O111, O128, and O157 or flagellar antigen of nine different H serogroups of E. coli: H2, H7, H8, H10, H11, H12, H18, H19, and H25. All patients had antibodies against the native antigen and/or the LPS of E. coli O157, but positive agglutination with H7 was observed only in one patient. In the VT-ELISA with plates coated with purified VT 1 or VT 2, antibody against VT 2 was observed in the sera of five of six patients examined, but none of the patients possessed VT 1 antibody. These results indicate that the causative pathogen in these eight hemolytic-uremic syndrome cases is likely to be VT-producing E. coli O157. The passive hemagglutination assay described here is a very sensitive, simple, and rapid method. This assay is highly recommended for the serological diagnosis of VT-producing E. coli infections, particularly in patients infected by serogroup O157 strains. Furthermore, the VT-ELISA is useful in studying the role of VT in hemolytic-uremic syndrome.
Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi children with diarrhea: a controlled study
Authors: Sack R B , Albert M J , Alam K , Neogi P K , Akbar M S
Published: January 2020

We undertook a controlled study of children younger than 5 years in Bangladesh to determine whether enterotoxigenic Bacteroides fragilis (ETBF) was associated with diarrhea in this population. ETBF was isolated from 22 (6.1%) of 358 patients and 5 (1.2%) of 425 controls (P = 0.0001). In children younger than 1 year, however, low isolation rates (2 to 3%) were found in both patients and controls. In children older than 1 year, the rates were significantly higher in children with diarrhea (16 [9%] of 177) than in controls (2 [1%] of 264; P = 0.00001). When children with mixed infections with other known diarrheal pathogens were removed, the differences in children older than 1 year were still significant (7 [4%] of 177 versus 2 [1%] of 264; P = 0.033). The syndrome associated with ETBF was secretory in nature, with watery diarrhea, and of mild severity. These epidemiological and clinical findings are similar to those from a previous study of White Mountain Apaches in the United States and are the first to suggest that ETBF may also be an important diarrheal pathogen in other geographic areas and in the developing world where diarrhea is highly endemic.
Clonal relationships among Escherichia coli serogroup O78 isolates from human and animal infections
Authors: Chérifi A , Contrepois M , Picard B , Goullet P , Orskov I , Orskov F
Published: January 2020

We investigated the clonal relationships among 63 Escherichia coli strains of antigen serogroup O78 isolated from infections in humans, cattle, sheep, pigs, and chickens. Both septicemic and enterotoxigenic isolates were included in the study. A main group of 55 E. coli strains consisting of 52 septicemic isolates and 3 human enterotoxigenic E. coli isolates were clustered in related clones. The remaining eight strains, four human and four animal isolates, were clonally heterogeneous. The main group of 55 clonally related strains included isolates from human and animal infections. This result indicates that animals are a possible source of serogroup O78 septicemic E. coli infections in humans.
Characteristics of CDC group 3 and group 5 coryneform bacteria isolated from clinical specimens and assignment to the genus Dermabacter
Authors: Funke G , Stubbs S , Pfyffer G E , Marchiani M , Collins M D
Published: January 2020

Over a 1-year period, 11 isolates (including 5 from blood cultures) of the recently described CDC group 3 and group 5 coryneform bacteria were derived from clinical specimens and compared with reference strains. Biochemical characteristics indicated a very close relationship between CDC group 3 and group 5 coryneform bacteria. The ability of CDC group 3 and the inability of CDC group 5 coryneform bacteria to ferment xylose were the only reactions that were different for the two taxa. Chemotaxonomic features of the two groups included the presence of meso-diaminopimelic acid, a lack of mycolic acids, and the presence of predominantly branched cellular fatty acids, a combination found among gram-positive rods only in Brevibacterium spp., Brachybacterium faecium, and Dermabacter hominis. 16S rRNA gene sequence analysis revealed that CDC group 3 and group 5 coryneform bacteria are members of the genus Dermabacter, which to date has been isolated exclusively from human skin.
Single-tube, noninterrupted reverse transcription-PCR for detection of infectious bursal disease virus
Authors: Lee L H , Ting L J , Shien J H , Shieh H K
Published: January 2020

An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used.
Comparison of Difco ESP and Organon Teknika BacT/Alert continuous-monitoring blood culture systems
Authors: Zwadyk P , Pierson C L , Young C
Published: January 2020

The Difco ESP and Organon Teknika BacT/Alert (BTA) systems were evaluated in a clinical study of 5,421 aerobic and 5,035 anaerobic blood cultures. Of 405 clinically significant positive cultures evaluated, 272 grew in both systems, 86 grew in ESP only, and 47 grew in BTA only (P < 0.005). Of 320 organisms detected in aerobic bottles, 208 grew in both systems, 68 grew in ESP only and 45 grew in BTA only (P < 0.05), with Staphylococcus aureus the only organism showing a statistically significant difference. The ESP anaerobic bottle also detected more anaerobes (16 of 17 versus 4 of 17, P < 0.005) and more organisms overall (57 versus 34, P < 0.05). However, with the exception of patients with anaerobic bacteremia (12 of 13 for ESP and 4 of 13 for BTA, P < 0.05), there was no statistical difference in the detection of patient episodes. Average detection time of matched aerobic bottles was 18.3 h for ESP and 22.0 h for BTA (P < 0.001). For matched pairs of anaerobic bottles, the average detection time was faster in the BTA bottles (P < 0.001), because of the growth of facultative organisms. To explore the differences in anaerobic detection more fully, 20 sets of anaerobic bottles were seeded with 12 anaerobic species mixed with human blood. ESP grew more organisms (17 of 20 versus 10 of 20, P < 0.025), and the average time to detection for the 10 paired positive cultures was 21.6 h for ESP and 50.8 h for BTA (P < 0.05). Times for loading and unloading bottles were similar for both systems.
Molecular epidemiology of ampicillin-resistant clinical isolates of Salmonella enteritidis
Authors: Vatopoulos A C , Mainas E , Balis E , Threlfall E J , Kanelopoulou M , Kalapothalki V , Malamou-Lada H , Legakis N J
Published: January 2020

During the last 6 years in Greece, there has been a significant increase in the number of ampicillin-resistant Salmonella clinical isolates reported. In this study 23 ampicillin-resistant Salmonella strains, consecutively isolated from patients with epidemiologically unrelated cases of food poisoning, were investigated. By serotyping and phage typing, 21 of these strains were identified as Salmonella enteritidis phage type 6a, 1 was identified as Salmonella typhimurium, and 1 was identified as Salmonella saintpaul. By plasmid pattern analysis, the 21 S. enteritidis strains were further differentiated into five groups. Group I consisted of 5 strains (carrying two plasmids of ca. 38 and 34 MDa), group II consisted of 10 strains (three plasmids of ca. 38, 34, and 2.5 MDa), group III consisted of 3 strains (four plasmids of ca. 38, 34, 15, and 2.5 MDa), group IV consisted of 1 strain (five plasmids of ca. 100, 38, 34, 24, and 15 MDa), and group V consisted of 2 strains (three plasmids of ca. 100, 38, and 24 MDa). Ampicillin resistance was easily transferred to Escherichia coli and was associated with the transfer of the 34-MDa plasmid, classified in the N incompatibility group for all strains of groups I to IV, and with the transfer of the 100-MDa plasmid for the group V strains. EcoRI restriction endonuclease digestions showed an extensive homology among the 34-MDa conjugative R plasmids. Hybridizations of the EcoRI restriction fragments of the 34-MDa plasmids with a TEM-type probe revealed the locus of the beta-lactamase gene to be situated on a ca. 6.6-MDa fragment, common in all plasmids. These results indicate that ampicillin resistance in Greece is due to the spread of a limited number of clones of S. enteritidis phage type 6A, carrying related 34-MDa R plasmids. Work is in progress to obtain a better understanding of ampicillin resistance in S. enteritidis.
Identification of human cytomegalovirus strain with immediate-early (IE) antigen-specific monoclonal antibody is prevented by point mutation in IE gene
Authors: Zipeto D , Sarasini A , Rossi F , Baldanti F , Revello M G , Milanesi G , Gerna G
Published: January 2020

In an AIDS patient with a disseminated human cytomegalovirus (HCMV) infection, presence of HCMV in blood was repeatedly excluded by the shell vial culture method with the HCMV immediate-early (IE) antigen-specific monoclonal antibody (MAb) 5D2 currently employed for rapid HCMV identification, whereas it was repeatedly confirmed by all other assays (conventional virus isolation from blood, antigenemia, and DNAemia). Sequence analysis of the HCMV strain revealed a point mutation in exon 2 of the IE gene, which led to a serine-to-proline substitution at position 20 of the corresponding protein. Cloning and expression of a region of the IE gene containing the mutation showed that this was responsible for the lack of reactivity of MAb 5D2. A pool of IE antigen-reactive MAbs instead of a single MAb must be used for rapid HCMV identification to detect all viral strains.
Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay
Authors: Gheesling L L , Carlone G M , Pais L B , Holder P F , Maslanka S E , Plikaytis B D , Achtman M , Densen P , Frasch C E , Käyhty H
Published: January 2020

A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre- and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre- and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the between-laboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 micrograms of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 micrograms of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.
Comparison of BACTEC Plus 26 and 27 media with and without fastidious organism supplement with conventional methods for culture of sterile body fluids
Authors: Fuller D D , Davis T E , Kibsey P C , Rosmus L , Ayers L W , Ott M , Saubolle M A , Sewell D L
Published: January 2020

We compared the BACTEC Plus 26/27 culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) with and without fastidious organism supplement with conventional centrifugation preparation and plating for the recovery and speed of detection of microorganisms. A total of 1,101 sterile body fluid specimens were collected and processed at five hospital laboratories, yielding 234 (21%) positive cultures. Of the 176 isolates considered clinically significant, 133 (76%) were recovered by both the BACTEC system and conventional culture, while 28 (16% [P < 0.005]) were recovered by BACTEC only and 11 (6%) were recovered by conventional culture alone. There were no statistically significant differences in the speed of detection of microbial growth. It was found that BACTEC, with or without the addition of fastidious organism supplement, exhibited improved sensitivity for the recovery of microorganisms, including fastidious bacteria.
Effect of centrifuging shell vials at 3,500 x g on detection of viruses in clinical specimens
Authors: Engler H D , Selepak S T
Published: January 2020

An increase in shell vial centrifugation force to 3,500 x g and a concomitant reduction in spin time to 15 min did not decrease the sensitivity of detecting viruses in clinical specimens compared with the accepted practice of using 700 x g for 40 min. No damage to the cell monolayer (ML) at the higher g force was observed. Toxicity to the ML is decreased with the shorter spin, probably because of reduced time of contact between the specimen and the ML.
Development and evaluation of rapid monoclonal antibody-based coagglutination test for direct detection of Vibrio cholerae O139 synonym Bengal in stool samples
Authors: Qadri F , Chowdhury A , Hossain J , Chowdhury K , Azim T , Shimada T , Islam K M , Sack R B , Albert M J
Published: January 2020

A monoclonal antibody-based coagglutination test directly detected Vibrio cholerae O139 synonym Bengal in 83 of 120 watery diarrheal stool specimens; on culture, 90 samples were positive. Thus, with 92% sensitivity, 100% specificity, and 100% positive and 95% negative predictive values, the coagglutination test is a useful rapid test for V. cholerae O139.
Use of arbitrary primer PCR to type Clostridium difficile and comparison of results with those by immunoblot typing
Authors: Killgore G E , Kato H
Published: January 2020

An arbitrarily primed PCR (AP-PCR) assay was used to type Clostridium difficile isolates from a hospital outbreak of antibiotic-associated diarrhea. Forty-one isolates were separated into nine groups, with 66% falling into one group; no other group contained more than 10%. Comparison of AP-PCR grouping with that when the immunoblot technique was used showed agreement for 33 of 34 isolates typed by both techniques, and AP-PCR grouped seven isolates that were not typeable by immunoblotting.
Comparison of nested PCR for detection of DNA in plasma with pp65 leukocytic antigenemia procedure for diagnosis of human cytomegalovirus infection
Authors: Freymuth F , Gennetay E , Petitjean J , Eugene G , Hurault de Ligny B , Ryckelynck J P , Legoff C , Hazera P , Bazin C
Published: January 2020

A nested PCR was used for the detection of human cytomegalovirus (HCMV) DNA in plasma. The presence of HCMV DNA and its correlation to pp65 leukocytic antigenemia were investigated with 299 blood samples from 45 organ transplant recipients and 63 AIDS patients. Of the 53 samples positive by nested PCR, 52 (98%) were also positive for leukocytic antigenemia and 23 had high levels of antigenemia (> 50 positive cells per 2 x 10(5) leukocytes). Of the 246 samples negative in PCR, only 3 (1.2%) had highly positive antigenemia. For 15 patients having a high antigenemia level in the course of their disease, consecutive blood samples were studied and also assessed for viremia in culture. The extent to which HCMV DNA, detected by PCR, was present in plasma correlated with increased levels of HCMV leukocytic antigenemia for six of the eight AIDS patients and for all the organ transplant recipients. Positivity for HCMV DNA in PCR and for viremia in cell culture was usually restricted to the highest antigenemia levels. From a total of 69 blood samples, PCR and culture gave positive results, respectively, for 17 of 32 samples (53%) and 14 of 32 samples (43%) from transplant recipients and for 15 of 37 samples (40%) and 9 of 37 samples (24%) from AIDS patients. Our findings have shown a strong correlation between high levels of leukocytic antigenemia and HCMV DNA in plasma. The detection of HCMV DNA in plasma by this nested PCR can prove HCMV dissemination in blood, but it lacks the rapidity and simplicity of the leukocytic pp65 antigenemia procedure.
Extensive DNA sequence conservation throughout the Mycobacterium tuberculosis complex
Authors: Frothingham R , Hills H G , Wilson K H
Published: January 2020

The Mycobacterium tuberculosis complex includes the four species M. tuberculosis, M. bovis, M. africanum, and M. microti. We sequenced 13 M. tuberculosis complex strains in the 16S-to-23S rDNA internal transcribed spacer (ITS). The ITS has a high rate of nucleotide substitution. Previous reports found three nucleotide substitutions in the ITS between two M. tuberculosis complex strains. In contrast, we found the same ITS sequence in all 13 M. tuberculosis complex strains (including all four species and M. bovis BCG). This finding confirms the conservation of 16S rDNA sequence and the high DNA-DNA relatedness found in previous studies. By the usual criteria, the four species of the M. tuberculosis complex would be considered a single species. In a phylogenetic analysis based on the ITS sequence, the four species of the M. tuberculosis complex were distinct from nontuberculous mycobacteria. The ITS contains at least seven potential sites for oligonucleotide probes with specificity for the M. tuberculosis complex.
Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis
Authors: Cameron D N , Khambaty F M , Wachsmuth I K , Tauxe R V , Barrett T J
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes). Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements. In general, strains which were different by MEE or ribotyping also had different PGFE patterns. PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping. All V. cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns. PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia. Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive. PFGE also distinguished nontoxigenic isolates endemic to the U.S. Gulf Coast from unrelated nontoxigenic isolates. In the present study PFGE was more discriminating than other previously described subtyping assays for V. cholerae O1 and appears to be a useful epidemiologic tool.
Clinical impact of rapid in vitro susceptibility testing and bacterial identification
Authors: Doern G V , Vautour R , Gaudet M , Levy B
Published: January 2020

During the past decade, a variety of instrument-assisted bacterial identification and antimicrobial susceptibility test systems have been developed which permit provision of test results in a matter of hours rather than days, as has been the case with traditional overnight procedures. These newer rapid techniques are much more expensive than older methods. It has been presumed but not proven that the clinical benefits of rapid testing to patients with infection offset the added cost. The intent of this study was to objectively define the clinical impact of rapid bacterial identification and antimicrobial susceptibility testing. A 1-year study was performed in which infected, hospitalized patients in a tertiary-care, teaching, medical center were randomly assigned to one of two groups: patients for whom identification and susceptibility testing was performed by using a semi-automated, rapid, same-day procedure and those for whom testing was accomplished by using traditional overnight techniques. The two groups were compared with respect to numerous demographic descriptors, and then patients were monitored prospectively through the end of their hospitalization with the aim of determining whether there existed objectively defineable differences in management and outcome between the two groups. The mean lengths of time to provision of susceptibility and identification test results in the rapid test group were 11.3 and 9.6 h, respectively. In the overnight test group, these values were 19.6 and 25.9 h, respectively (P < 0.0005). There were 273 evaluable patients in the first group and 300 in the second group. Other than the length of time required to provide susceptibility and identification test results, no significant differences were noted between the two groups with respect to > 100 demographic descriptors. With regard to measures of outcome, the mean lengths of hospitalization were also the same in both groups. Mortality rates were however, lower in the rapid test group (i.e., 8.8% versus 15.3%). Similarly, statistically significantly fewer laboratory studies, imaging procedures, days of intubation, and days in an intensive or intermediate-care area were observed with patients in the rapid test group. Rapid testing was also associated with significantly shortened lengths of elapsed time prior to alterations in antimicrobial therapy. Lastly, patient costs for hospitalization were significantly lower in the rapid test group. The results of this study indicate the rapid same-day bacterial identification and susceptibility testing in the microbiology laboratory can have a major impact on the care and outcome of hospitalized patients with infection.
Comparison of two methods for detecting intrathecal synthesis of Borrelia burgdorferi-specific antibodies and PCR for diagnosis of Lyme neuroborreliosis
Authors: Zbinden R , Goldenberger D , Lucchini G M , Altwegg M
Published: January 2020

An indirect immunofluorescence assay (IFA), a commercial capture enzyme-linked immunosorbent assay (ELISA), and PCR were compared for the diagnosis of Lyme neuroborreliosis in 16 individuals. Borrelia burgdorferi-specific intrathecal immunoglobulin G was found in 10 of 16 patients by IFA and in 8 of 12 patients by capture ELISA. PCRs performed on cerebrospinal fluid samples stored at -70 degrees C were positive for one of eight children and one of four adults with intrathecal antibody production and negative for all four adults without intrathecal antibody production. For two children with facial palsy following erythema chronicum migrans, neither IFA nor capture ELISA detected specific intrathecal antibodies and PCR was also negative. We conclude that both clinical manifestations and laboratory parameters are important for the diagnosis of Lyme neuroborreliosis.
Comparison of four different methods for epidemiologic typing of Acinetobacter baumannii
Authors: Seifert H , Schulze A , Baginski R , Pulverer G
Published: January 2020

A set of 103 epidemiologically well-defined Acinetobacter baumannii isolates obtained from nine hospital outbreaks and 21 unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) of total genomic DNA digested with ApaI. Among outbreak strains, eight different patterns and five possible variants were identified by PFGE. Results were compared with those from traditional typing methods such as plasmid profile analysis, antimicrobial susceptibility, and biotyping. Plasmid analysis revealed six different and two related patterns; one outbreak strain lacked plasmids. A total of 16 of the 21 unrelated strains harbored plasmids and exhibited unique patterns. Epidemiologically unrelated strains were placed into only two biotypes and had similar antimicrobial susceptibility patterns but were clearly distinguished by PFGE. PFGE of A. baumannii chromosomal DNA yielded reproducible and easily readable results and showed excellent discriminatory power. However, plasmid profile analysis may provide a cost-effective first step in epidemiological typing of A. baumannii isolates obtained from well-defined hospital outbreaks.
Improved enrichment broth for cultivation of fastidious organisms
Authors: Cartwright C P , Stock F , Gill V J
Published: January 2020

An enrichment broth developed in our laboratory, fastidious broth (FB), was compared with two commercially available broth media, supplemented thioglycolate broth and enriched eugonic broth. FB supported the growth of a number of organisms that were not cultivatable in either of the other two media, including Corynebacterium jeikeium, Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae. In addition, for several organisms that were able to grow in all three broths, including Neisseria meningitidis, Nocardia asteroides, and Actinomyces spp., both the time of incubation and the starting inoculum necessary to enable detection of growth were decreased significantly by using FB.
Random amplified polymorphic DNA genotyping of Mycobacterium malmoense
Authors: Kauppinen J , Mäntyjärvi R , Katila M L
Published: January 2020

The suitability of random amplified polymorphic DNA-PCR for the detection of differences between Mycobacterium malmoense strains was evaluated. With two of the 32 tested primers seven fingerprint patterns which proved excellent in distinguishing intraspecies variations of M. malmoense were obtained. The combination of the results obtained with the two primers permitted a clear separation of the strains. This technique is useful for analyzing species whose DNA sequences are not known. It can easily be adapted to test any mycobacterial species.
Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep
Authors: Blasco J M , Marín C , Jiménez de Bagués M , Barberán M , Hernández A , Molina L , Velasco J , Díaz R , Moriyón I
Published: January 2020

A total of 291 unvaccinated sheep from Brucella melitenesis-infected flocks were examined for delayed-type hypersensitivity (DTH) responses with Brucellergene commercial allergen and with cold saline extract and cytosol from rough B. melitensis 115, and their sera were tested in the rose bengal test (RBT), complement fixation test (CFT), and enzyme-linked immunosorbent assay (ELISA) with lipopolysaccharide. DTH reactions were maximal after 72 h, with no intensity differences among allergens, inoculation sites (eyelid and tail), and doses tested. There were no differences in the results recorded by visual inspection and palpation of inoculation sites, by measuring skin thickness with a caliper, or by microscopic examination of samples taken at necropsy. Six days after DTH testing, energy was observed in 100% of the animals, and 100% reactivity was recovered only after 24 days. All animals were necropsied, and thorough bacteriological searches were performed. The sensitivities found with the 140 animals from which B. melitensis was isolated were ELISA, 100%; DTH, 97.1%; RBT, 92.1%; and CFT, 88.6%. Those results put into question the value of RBT and CFT as screening and confirmatory tests for sheep brucellosis and at least indicate that their standardization should be modified. For 151 tested sheep from which B. melitensis was not isolated, the percentages of positive animals were ELISA, 100%; DTH, 94.0%; RBT, 57.6%; and CFT, 53.6%. All tests were negative for 100 tested sheep from Brucella-free flocks. The different results of bacteriological and immunological tests suggest the usefulness of developing indirect tests able to distinguish truly infected animals from those that have developed an immunological response.
Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics and antimicrobial susceptibilities
Authors: Riegel P , de Briel D , Prévost G , Jehl F , Monteil H
Published: January 2020

Levels of DNA relatedness were determined by performing DNA-DNA hybridization experiments (S1 nuclease procedure) with 13 human isolates exhibiting various antimicrobial susceptibility patterns which had been identified as Corynebacterium jeikeium by classical tests and the API Coryne system and with reference strains of C. jeikeium and related taxa. Twelve of 13 isolates which formed three genomic groups showed between 22 and 75% relatedness with the type strain of C. jeikeium. One of these genomic groups included all the strains resistant to penicillin and gentamicin and is genomically related to the C. jeikeium type strain at the species level. In addition, the reference strain of "Corynebacterium genitalium" biotype II was found to belong to this genospecies and therefore can be considered as a synonym of C. jeikeium. In contrast, one isolate and the reference strains of "Corynebacterium pseudogenitalium" biotypes C-3 and C-4 which were assigned to C. jeikeium by the API Coryne system were less than 10% related to the C. jeikeium type strain. These nongenomically related strains can be differentiated from the jeikeium-related strains on the basis of positive acidification from fructose and growth under anaerobic conditions. Furthermore, these strains exhibited full susceptibility to penicillin whereas the strains related to the C. jeikeium type strain are resistant to or only moderately susceptible to penicillin. No genomic relationship was found between C. jeikeium-related strains and other lipophilic coryneforms, identified as Corynebacterium accolens or Corynebacterium group G or F. Our study demonstrates the necessity to perform the fructose fermentation test or respiratory-type test for the correct identification of lipophilic coryneforms as C. jeikeium. Although these strains show genomic diversity at the species level, in a practical aspect, biochemical properties as well as antimicrobial susceptibility may allow the classification of such isolates in this single taxon.
Recognition of Dermabacter hominis, formerly CDC fermentative coryneform group 3 and group 5, as a potential human pathogen
Authors: Gruner E , Steigerwalt A G , Hollis D G , Weyant R S , Weaver R E , Moss C W , Daneshvar M , Brenner D J
Published: January 2020

Thirty strains of fermentative coryneform-like bacteria designated CDC fermentative coryneform group 3 and coryneform group 5 were compared biochemically by cellular fatty acid analysis and by DNA relatedness with the type strain of Dermabacter hominis, ATCC 49369. DNA from 22 strains of both CDC groups showed 69 to 96% relatedness (hydroxyapatite method) to labeled DNA from ATCC 49369 and to DNA from CDC group 3 strain G4964, and the strains are considered to belong to D. hominis. The remaining eight strains were genetically but not phenotypically differentiable from D. hominis. They were genetically heterogeneous, but hybridization results indicated that they probably belong to the genus Dermabacter. Thirteen of the 22 D. hominis strains and all 8 of the other Dermabacter strains had been isolated from blood, which indicates the pathogenic potential of this species and genus.