bluuebunny/crossref_metadata_2025 huggingface dataset:
Patient-to-patient spread of a single strain of Corynebacterium striatum causing infections in a surgical intensive care unit
Authors: Brandenburg A H , van Belkum A , van Pelt C , Bruining H A , Mouton J W , Verbrugh H A
Published: January 2020

Over a 12-month period, Corynebacterium striatum strains were isolated from clinical specimens from 14 patients admitted to a surgical intensive care unit. These isolates were identical by morphology and biotype and displayed the same antibiogram. Ten isolates were found to be the sole possible pathogen. These 10 isolates were from six patients, three of whom had signs of infection at the time of positive culture. Further typing was performed by random amplification of polymorphic DNA analysis, by which all strains were identical and were found to differ to various degrees from reference strains and from isolates found in clinical samples from other wards. In a case-control study the only independent risk factor for acquiring the strain was intubation for longer than 24 h (odds ratio, 20.09; 95% confidence interval, 2.29 to 176.09). The same strain was isolated from surfaces and from air sampled in the direct vicinity of infected patients but never from surfaces or air in other places of the ward. The strain was not isolated from the ventilators. The strain was cultured from the hands of personnel attending to infected patients, but no long-term carriers were found among members of the hospital personnel, suggesting transient carriage only. We conclude that C. striatum can cause serious nosocomial infections in surgical intensive care unit patients and may spread from patient to patient via the hands of attending personnel.
Heterogeneity in susceptibility to metronidazole among Helicobacter pylori isolates from patients with gastritis or peptic ulcer disease
Authors: Weel J F , van der Hulst R W , Gerrits Y , Tytgat G N , van der Ende A , Dankert J
Published: January 2020

Combination therapies that include metronidazole (MTZ) are the most successful therapies used in eradicating Helicobacter pylori. In this study, the prevalence and the relevance of heterogeneity in susceptibility to MTZ among H. pylori populations of 156 patients were evaluated. The results of this study show that 37 patients (24%) were infected with MTZ-resistant H. pylori (MIC > or = 8 micrograms/ml). Furthermore, 33% (52 of 156) of the patients were found to be infected with H. pylori populations heterogeneous for their susceptibility to MTZ. The reassessment of the MICs of MTZ for these 52 H. pylori populations revealed MTZ resistance in 28 of them, increasing the number of MTZ-resistant H. pylori populations among the 156 patients to 65 (42%). Out of 20 isolates, 2 (10%) heterogeneous in their susceptibility to MTZ also appeared to be heterogeneous at the genome level as determined by randomly amplified polymorphic DNA fingerprinting. In conclusion, the results show the limitations and risk of possible misinterpretations when only a single colony, picked from the primary H. pylori populations isolated from patients, is analyzed for its susceptibility to MTZ.
Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit
Authors: Arpin C , Coze C , Rogues A M , Gachie J P , Bebear C , Quentin C
Published: January 2020

In 1993, 63 isolates of Enterobacter aerogenes were collected from 41 patients in a medical intensive care unit (ICU). During the same period, only 46 isolates from 32 patients were collected in the rest of the hospital. All isolates were analyzed by antibiotic resistance phenotype, and 77 representative isolates were differentiated by plasmid restriction analysis, ribotyping, and arbitrarily primed (AP)-PCR. The extended-spectrum beta-lactamases produced by 22 strains were characterized by determination of their isoelectric points and by hybridization of plasmid DNA with specific probes. The isolates were divided into 25 antibiotic resistance phenotypes, either susceptible (group I) or resistant (group II) to aminoglycosides, and exhibited three phenotypes of resistance to beta-lactams: chromosomally derepressed cephalosporinase alone or associated with either extended-spectrum beta-lactamases (mainly of the SHV-4 type) or imipenem resistance. The results of the tests divided the 77 representative isolates (group I, n = 21; group II, n = 56) into 15 plasmid profiles, 14 ribotypes, and 15 AP-PCR patterns. Although the resistant isolates (group II) exhibited different plasmid profiles, ribotyping and AP-PCR analysis demonstrated an identical chromosomal pattern, indicating an epidemiological relatedness. They were mainly found in the medical ICU and occasionally in other units. The susceptible strains (group I) had various and distinct markers and were mainly isolated in units other than the medical ICU. In conclusion, the presence of a nosocomial outbreak in an ICU and the spread of a multidrug-resistant epidemic strain throughout the hospital was confirmed. Ribotyping and AP-PCR represent discriminatory tools for the investigation of nosocomial outbreaks caused by E. aerogenes.
Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopolysaccharide
Authors: Simoons-Smit I M , Appelmelk B J , Verboom T , Negrini R , Penner J L , Aspinall G O , Moran A P , Fei S F , Shi B S , Rudnica W , Savio A , de Graaff J
Published: January 2020

Recently, it has been shown that the lipopolysaccharide (LPS) O antigen of Helicobacter pylori contains Lewis x (Lex), Lewis y (Ley), or both Lex and Ley antigens. We applied a serotyping method for H. pylori by an enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) specific for these antigens and the related fucosylated H type 1 (H1) antigen. The selected MAbs recognized the Lex and/or Ley structures in the LPS of H. pylori. The agreement between the results of biochemical compositional analysis and the serological data validated our serotyping system. A total of 152 strains from different geographic origins (The Netherlands, Canada, Poland, Italy, and People's Republic of China) were examined for typeability based on the presence of Lewis antigens. One hundred twenty-nine (84.9%) strains were typeable, and 12 different serotyping patterns were observed; 80.9% of the strains contained Lex and/or Le(y) antigens, and 18.4% reacted with the MAb against the related H1 antigen either alone or in combination with the Lex and/or Ley antigen. Our results show that the Lex and Ley antigens are frequently encountered in the LPS of H. pylori strains from various geographic origins. This typing method is an easy-to-perform technique, which can be used for strain differentiation in epidemiological studies of H. pylori infections.
Comparison of five tests for identification of Staphylococcus aureus from clinical samples
Authors: Luijendijk A , van Belkum A , Verbrugh H , Kluytmans J
Published: January 2020

Five different laboratory tests for the identification of Staphylococcus aureus were compared. Analyses of 271 presumptive S. aureus strains, supplemented with 59 well-defined methicillin-resistant S. aureus (MRSA) isolates, were performed. Only the Staphaurex Plus (Murex Diagnostics, Dartford, United Kingdom) and the Pastorex Staphplus (Sanofi, Marnes-La-Coquette, France) tests displayed 100% sensitivity. The observed difference with the free-coagulase test (Bacto coagulase plasma; Difco, Detroit, Mich.), a bound-coagulase (clumping factor) test, and the former Staphaurex test (Murex Diagnostics) was caused mainly by the inability of these three tests to identify some MRSA strains correctly. Among Polish MRSA isolates included in the analysis, a group of free-coagulase-negative S. aureus strains was detected. Genetic typing by random amplification of polymorphic DNA revealed that the strains showing aberrant behavior when the different test results were compared belonged to limited number of S. aureus clones.
Isolation of an unusual mycobacterium from an AIDS patient
Authors: Tortoli E , Kirschner P , Bartoloni A , Burrini C , Manfrin V , Mantella A , Scagnelli M , Scarparo C , Simonetti M T , Böttger E C
Published: January 2020

A mycobacterium isolated from a clinical sample of an AIDS patient was identified as Mycobacterium interjectum by direct 16S rRNA sequence determination. High-performance liquid chromatography, however, revealed a mycolic acid pattern which was different from the one shared by the previously analyzed strains of this species.
Pulmonary paracoccidioidomycosis misdiagnosed as Pneumocystis pneumonia in an immunocompromised host
Authors: Silletti R P , Glezerov V , Schwartz I S
Published: January 2020

Yeast cells of Paracoccidioides brasiliensis can resemble the cysts of Pneumocystis carinii in smears stained with Grocott's modification of the Gomori methanamine silver stain. Furthermore, P. brasiliensis can cross-react in material stained with a widely used P. carinii immunofluorescent stain which uses monoclonal antibodies. The need to differentiate P. brasiliensis and P. carinii will become more important as the increasing incidence of immunosuppression results in the reactivation of latent P. brasiliensis infections.
The degrees of hepatocyte nuclear but not cytoplasmic expression of hepatitis B core antigen reflect the level of viral replication in chronic hepatitis B virus infection
Authors: Chu C M , Yeh C T , Chien R N , Sheen I S , Liaw Y F
Published: January 2020

Although immunodetection of hepatitis B core antigen (HBcAg) in the liver has long been recognized as a marker of active hepatitis B virus (HBV) replication, the correlation between the level of viral replication and the degrees of expression of HBcAg in hepatocytes remains to be established. In this study, we examined the semiquantitative relationship between the level of HBV DNA in serum and the degree of expression of HBcAg in the hepatocyte nucleus or cytoplasm in 80 adults with chronic hepatitis B. Expression of HBcAg in hepatocytes was studied by the avidin-biotin immunoperoxidase method, and the results were scored on a scale of 0 to 4, values corresponding to the positivity of 0, 1 to 10, 11 to 25, 26 to 50, and > 50%, respectively, of hepatocytes examined. Serum HBV DNA was tested by a liquid hybridization assay, and the results were scored on a scale of 0 to 5, values corresponding to undetectable levels and levels of < or = 50, 51 to 100, 101 to 150, 151 to 200, and > 200 pg/ml, respectively. The results revealed a highly significant, positive correlation between the level of HBV DNA in serum and the degree of expression of HBcAg in nuclei (Spearman rank correlation coefficient [rs] = 0.653, P < 0.001). The mean scores (95% confidence intervals) of levels of HBV DNA in sera of patients whose levels of expression of HBcAg in nuclei received a score of 0 (n = 33), 1 or 2 (n = 35), and 3 or 4 (n = 12) were 1.3 (1.1 to 1.5), 2.5 (2.1 to 2.9), and 3.9 (3.1 to 4.7), respectively. However, no correlation between the level of HBV DNA in serum and the degree of expression of HBcAg in the cytoplasm was noted (rs = 0.026, P > 0.8). In conclusion, the degree of expression of HBcAg in the hepatocyte nucleus but not the cytoplasm can accurately reflect the level of viral replication in patients with chronic hepatitis B.
Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge
Authors: Shaio M F , Lin P R , Liu J Y
Published: January 2020

A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.
Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi
Authors: Espinel-Ingroff A , Bartlett M , Bowden R , Chin N X , Cooper C , Fothergill A , McGinnis M R , Menezes P , Messer S A , Nelson P W , Odds F C , Pasarell L , Peter J , Pfaller M A , Rex J H , Rinaldi M G , Shankland G S , Walsh T J , Weitzman I
Published: January 2020

A multicenter study was conducted to expand the generation and analysis of data that supports the proposal of a reference method for the antifungal susceptibility testing of filamentous fungi. Broth microdilution MICs of amphotericin B and itraconazole were determined in 11 centers against 30 coded duplicate pairs of Aspergillus spp., Fusarium spp., Pseudallescheria boydii, and Rhizopus arrhizus. The effect of inoculum density (approximately 10(3) and 10(4) CFU/ml), incubation time (24, 48, and 72 h), and procedure of MIC determination (conventional and colorimetric [Alamar Blue] evaluation of growth inhibition) on intra- and interlaboratory agreement was analyzed. Based on intra- (97 to 100%) and interlaboratory (94 to 95%) agreement for both drugs, the overall optimal testing conditions identified were determination of colorimetric MICs after 48 to 72 h of incubation with an inoculum density of approximately 10(4) CFU/ml. These testing conditions are proposed as guidelines for a reference broth microdilution method.
Comparison of five agglutination tests for identification of Staphylococcus aureus
Authors: Wilkerson M , McAllister S , Miller J M , Heiter B J , Bourbeau P P
Published: January 2020

Various commercially produced agglutination kits are widely used for the identification of Staphylococcus aureus. These kits detect the presence of protein A and/or clumping factor on S. aureus. The literature has shown that methicillin-resistant S. aureus (MRSA) isolates which are deficient in both clumping factor and protein A may be misidentified. Two products, Slidex and Staphaurex Plus, utilize specific anti-S. aureus antibodies, potentially giving them greater sensitivity compared to products without these antibodies. We report a prospective study designed to compare the performance characteristics of Fastaph, Slidex, Staphaurex, Staphaurex Plus, Staphyloslide, and the tube coagulase test for the identification of staphylococcal isolates. All discrepant isolates were tested with the Gen-Probe AccuProbe S. aureus test and were identified to the species level with conventional reference biochemicals. A total of 1,193 isolates were tested, including 33 MRSA and 423 methicillin-sensitive S. aureus isolates. The sensitivities and specificities of the tests, respectively, were as follows: Fastaph, 99.1 and 98.9%; Slidex, 99.6 and 96.4%; Staphaurex, 98.9 and 99.9%; Staphaurex Plus, 99.6 and 93.9%; Staphyloslide, 99.1 and 98.9%; and tube coagulase, 99.3 and 100%. Sensitivity was excellent for all of the products tested. The specificities of Fastaph, Staphaurex, and Staphyloslide were excellent, while Staphaurex Plus and Slidex demonstrated less optimal results.
False-positive results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test
Authors: Liesenfeld O , Press C , Montoya J G , Gill R , Isaac-Renton J L , Hedman K , Remington J S
Published: January 2020

Although tests for detection of immunoglobulin M (IgM) toxoplasma antibodies have been reported to have a high degree of accuracy, it is well recognized by investigators in the United States and Europe that false-positive results may occur with many of these tests, at times to an alarming degree. Unfortunately, this information is not well documented in the literature. Studies on various toxoplasma IgM test kits are frequently flawed. The investigators often use reference tests which have not previously been carefully evaluated as well as sera that were not appropriate to answer the question of how often false-positive results might occur. We recently had the unique opportunity to evaluate the accuracy of the Platelia Toxo IgM test in 575 serum samples obtained during an outbreak of toxoplasmosis which occurred in 1995 in the Capital Regional District of British Columbia, Canada. When compared with results obtained in a reference IgM enzyme-linked immunosorbent assay (ELISA), the Platelia Toxo IgM test had a sensitivity of 99.4%, specificity of 49.2%, positive predictive value of 51.9%, negative predictive value of 99.3%, and an overall agreement of 67.0%. In an attempt to resolve discrepancies between these two tests, a serological profile (Sabin-Feldman dye test, IgA and IgE antibody tests, differential agglutination [AC/HS] test, and IgG avidity method) was performed. Of 153 serum samples that were positive in the Platelia Toxo IgM test and negative in the IgM ELISA, 71 (46.4%) were negative in the Sabin-Feldman dye test. Of the serum samples that were positive in the dye test, 77 (93.9%) had a serological profile most compatible with an infection acquired in the distant past. These results reveal high numbers of false-positive results in the Platelia Toxo IgM test and highlight the importance of appropriate evaluation of commercial tests that are currently being marked. Our results also emphasize the importance of confirmatory testing to determine whether the results of an IgM antibody test reflect the likelihood of a recently acquired infection.
High rates of genital mycoplasma infection in the highlands of Papua New Guinea determined both by culture and by a commercial detection kit
Authors: Clegg A , Passey M , Yoannes M , Michael A
Published: January 2020

Duplicate vaginal swabs were collected from 100 women, and comparisons were made between an in-house broth-agar culture system and a commercially available kit, the Mycoplasma IST kit (bioMérieux), for the detection of Mycoplasma hominis and Ureaplasma urealyticum. There was good agreement between the two systems for detection of the genital mycoplasmas in terms of sensitivity, with values of > 92% being obtained. In terms of specificity, the mutual comparisons were less favorable, though specificity values of > 72% were obtained. Statistically there was no significant difference in the performance of the two tests (P < 0.1 for both M. hominis and U. urealyticum). While the broth-agar culture system was considerably less expensive than the kit, the Mycoplasma IST kit provided additional information on antibiotic susceptibilities and had the advantages of a shelf life of up to 12 months and not requiring the preparation of culture media. The prevalences of colonization obtained for M. hominis and U. urealyticum were extremely high in this randomly selected group of women from periurban and rural settlements in the Eastern Highlands of Papua New Guinea, being > or = 70% for M. hominis and > or = 78% for U. urealyticum. colonization with both genital mycoplasmas simultaneously was also very common, with > or = 60% of women being colonized by both M. hominis and U. urealyticum.
Molecular characterization of Escherichia coli strains isolated from pigs with edema disease
Authors: Aarestrup F M , Jorsal S E , Ahrens P , Jensen N E , Meyling A
Published: January 2020

The present study was conducted to investigate the epidemiological relationship of isolates of Escherichia coli causing edema disease. Classical edema disease has not previously been described in Denmark, but between February 1994 and November 1995 cases appeared in 51 pig herds, among which direct or indirect trading contacts were confirmed for 36 of the herds. A total of 213 isolates from pigs with edema disease in Denmark and other countries and 23 E. coli O139 isolates from pigs with diarrhea or healthy pigs were analyzed to characterize their O serogroups, HindIII ribotypes, and pulsed-field gel electrophoresis (PFGE) types, and 183 of the isolates were also analyzed for their plasmid profiles. The resulting PFGE types of the isolates from pigs with edema disease were examined by cluster analysis. Ten isolates from three herds could not be typed with the available O antisera, whereas all other isolates were of serotype O139. However, all isolates from pigs with edema disease belonged to the same HindIII ribotype, which was not observed among the isolates from pigs with diarrhea or healthy pigs. All isolates from Danish pigs with edema disease except for three isolates originating from two herds belonged to the same or closely related XbaI PFGE types; the other three isolates were assigned to possibly related types. Isolates from pigs with edema disease in different countries belonged to different PFGE types. All isolates from Danish pigs with edema disease grouped together in one cluster, in contrast to isolates from other countries, which did not form any clusters. E. coli strains of serogroup O139 from pigs with diarrhea or isolated from the feces of healthy Danish pigs were very different. Plasmid profiles differed largely among isolates. However, among the isolates from Danish pigs with edema disease, one type predominated within herds. The present study indicated that most, if not all, of the observed cases of edema disease in Denmark were part of the same outbreak. The combination of PFGE typing and ribotyping was useful for studying the possible clonal relationship among strains, whereas plasmid profiling was less informative.
Rapid identification of pathogenic Leptospira species (Leptospira interrogans, L. borgpetersenii, and L. kirschneri) with species-specific DNA probes produced by arbitrarily primed PCR
Authors: Letocart M , Baranton G , Perolat P
Published: January 2020

Arbitrarily primed PCR (AP-PCR) assays can be used to discriminate between species of Leptospira. Comparative analysis of the fingerprints obtained from representative sets of serovar reference strains of Leptospira interrogans sensu stricto, L. borgpetersenii, and L. kirschneri and the reference strains of the other Leptospira spp. revealed species-specific DNA fragments. These species-specific sequences were reamplified in order to produce digoxigenin-11-dUTP-labeled genomic DNA probes that could be used to identify Leptospira species. Three probes (specific for L. interrogans sensu stricto, L. borgpetersenii, and L. kirschneri) were selected and tested with 72 representative serovar reference strains, all of which had previously been studied by DNA-DNA hybridization. The two techniques were in general agreement, and hybridization with AP-PCR-derived probes was shown to be a useful approach for rapid species determination of leptospires, without the prior need for DNA sequence information. These nonradioactive probes can be used to identify Leptospira species in nonspecialized laboratories, and this should contribute to a better knowledge of the molecular epidemiology of leptospirosis.
Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay
Authors: Gilbert S A , Larochelle R , Magar R , Cho H J , Deregt D
Published: January 2020

A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.
Lot-to-lot variability of antibiotic medium 3 used for testing susceptibility of Candida isolates to amphotericin B
Authors: Lozano-Chiu M , Nelson P W , Lancaster M , Pfaller M A , Rex J H
Published: January 2020

We have previously shown that use of antibiotic medium 3 in the microdilution variant of the M27-T test method permits detection of amphotericin B-resistant Candida isolates. As this medium is not standardized and our initial work used only a single lot of antibiotic medium 3, we studied the lot-to-lot variability of three commercial lots of antibiotic medium 3 obtained from two sources. The MICs obtained with the new lots were lower than those of with the original lot, but the new lots still consistently separated putatively resistant and putatively susceptible isolates and this permits proposal of possible breakpoints for this assay system.
Serological and bacteriological study of swine brucellosis
Authors: Lord V R , Cherwonogrodzky J W , Marcano M J , Melendez G
Published: January 2020

A serological and bacteriological study was performed with sera taken from 2,228 swine from six states in Venezuela. None of the animals were vaccinated against brucellosis, and the prevalence of the disease varied from 5 to 89% on farms located in these states. Our studies indicated that the animals could be categorized into four groups depending on the degree of reactivity in serological tests. Brucella suis biovar 1 was isolated from the lymph nodes, spleens, and semen samples of seropositive animals and identified by oxidative metabolic techniques. B. suis could not be isolated from tissues of seronegative swine even from farms with cases of the disease (detected by serology). Results suggest that, although the immunodiffusion assay using Brucella melitensis B115 polysaccharide B or B. abortus 1119-3 O-polysaccharide could be useful in the detection of active infections, it is perhaps not as sensitive as some of the other standard serological tests used in this study for the detection of swine brucellosis.
Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates
Authors: Milane G , Kourtesis A B , Dea S
Published: January 2020

Seven hybridoma cell lines producing monoclonal antibodies (MAbs) to the hemagglutinin-esterase (HE) glycoprotein of bovine coronavirus (BCV) were obtained from BALB/c mice that were immunized with an enriched peplomeric fraction of the winter dysentery (WD)-associated strain BCQ.2590. The specificities of these MAbs to either the dimeric (140-kDa) or the monomeric (65-kDa) form of the HE glycoprotein were determined by Western immunoblotting experiments with purified virus and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts. Four of these anti-HE MAbs inhibited the hemagglutinating activity of the virus and three weakly neutralized its infectivity in vitro. In addition, competition binding assays allowed for the definition of two independent antigenic domains (domains A and D) and two overlapping antigenic domains (domains B and C) for the HE glycoprotein of the WD-associated strain; epitopes located within antigenic domain A were not associated with hemagglutination inhibition (HAI) and virus neutralization activities. In HAI tests, the four anti-HA MAbs defined two distinct antigenic subgroups among 24 BCV field isolates that have been associated with either typical outbreaks of WD or neonatal calf diarrhea (NCD) in Quebec dairy herds from 1986 to 1996. The Quebec WD-associated strains of BCV, as well as some of the NCD-associated strains isolated since 1991, fell within a subgroup distinct from that of the prototype Mebus strain.
Rapid susceptibility testing of fungi by flow cytometry using vital staining
Authors: Wenisch C , Linnau K F , Parschalk B , Zedtwitz-Liebenstein K , Georgopoulos A
Published: January 2020

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined.
Characterization of and application of monoclonal antibodies against Rickettsia africae, a newly recognized species of spotted fever group rickettsia
Authors: Xu W , Beati L , Raoult D
Published: January 2020

Rickettsia africae is a newly described species which causes African tick bite fever. Mediterranean spotted fever caused by R. conorii is endemic in the same regions of Africa as tick bite fever, and differentiation of the two syndromes by characterization of their etiological agents is important for epidemiological studies. R. africae and R. conorii are, however, difficult to distinguish, and therefore, our aim was to produce monoclonal antibodies to address this problem. Monoclonal antibodies were produced against R. africae by fusing splenocytes from BALB/C mice immunized with purified rickettsial organisms and SP2/0-Ag14 myeloma cells. A total of 355 hybridomas producing monoclonal antibodies to R. africae were identified by initial screening with six different antigens by microimmunofluorescence assay. A panel of 23 representative monoclonal antibodies were selected and subcloned. This panel was screened with a further 17 different spotted fever group (SFG) rickettsial reference antigens. Of these 23 monoclonal antibodies, 1 cross-reacted with only R. parkeri, whereas the others cross-reacted with more than two different antigens. Immunoblotting indicated that all the monoclonal antibodies were directed against the epitopes on two major high-molecular-mass heat-labile proteins, of which the molecular masses were 128 and 135 kDa, respectively. This monoclonal antibody panel was used successfully to identify R. africae in the blood culture of an infected patient, in infected cells within shell vials, and in infected ticks collected from Africa. Furthermore, the cross-reactivity of each SFG rickettsia with each of these 23 monoclonal antibodies was scored and was used to build a dendrogram of taxonomic relatedness between R. africae and the other SFG rickettsiae on the basis of Jaccard coefficients and unweighted pair group method with arithmetic mean analysis. The relatedness was generally consistent with that obtained by other methods of comparison.
Growth characteristics and a novel method for identification (the WEE-TAB system) of Porphyromonas species isolated from infected dog and cat bite wounds in humans
Authors: Hudspeth M K , Hunt Gerardo S , Citron D M , Goldstein E J
Published: January 2020

Thirty-nine clinical isolates of Porphyromonas species recovered from infected cat and dog bite wounds in humans and eight American Type Culture Collection and National Collection of Type Cultures type strains were characterized by using the API ZYM system, the RapID ANA II system, and conventional biochemical methods. Growth characteristics on various agar media were compared. All strains grew on brucella blood agar supplemented with vitamin K1 and hemin and on brucella laked blood agar supplemented with vitamin K1 and hemin. In contrast, only 34% of strains grew on unsupplemented brucella blood agar, 62% grew on Columbia blood agar, and 70% grew on tryptic soy blood agar (the last three media did not contain vitamin K1 or hemin). The ability of the single-tube, triple-substrate WEE-TAB system to detect the preformed enzymes N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-fucosidase, trypsin-like activity, and chymotrypsin was evaluated. The WEE-TAB test results were easy to interpret; the WEE-TAB tests were more sensitive than the comparable tests with the API ZYM and RapID ANA II systems for the detection of alpha-D-galactosidase, beta-D-galactosidase, trypsin, and chymotrypsin, and the WEE-TAB tests accurately identified Porphyromonas species.
Molecular epidemiology of two international sprout-borne Salmonella outbreaks
Authors: Puohiniemi R , Heiskanen T , Siitonen A
Published: January 2020

Sprout-borne Salmonella outbreaks in Finland have increased during the last 10 years. The latest two were caused by Salmonella enterica serovar Bovismorbificans (antigenic structure 6,8:r:1,5) in 1994 and S. enterica serovar Stanley (4,5, 12:d:1,2) in 1995. In this study, the restriction fragment length polymorphism of genomic DNA after pulsed-field gel electrophoresis (PFGE) and antimicrobial resistance profiles of the outbreak and nonoutbreak strains were compared. In each separate outbreak, the PFGE patterns of the outbreak strains (40 strains of S. enterica serovar Bovismorbificans and 28 strains of S. enterica serovar Stanley) after digestion of genomic DNA with restriction enzyme XbaI were indistinguishable from each other but differed clearly from those of the nonoutbreak strains (26 strains of S. enterica serovar Bovismorbificans and 40 strains of S. enterica serovar Stanley). The restriction enzyme XhoI did not differentiate the outbreak and nonoutbreak strains. The S. enterica serovar Stanley strains associated with the outbreak also had a unique antimicrobial resistance pattern, whereas all S. enterica serovar Bovismorbificans strains, both outbreak and nonoutbreak strains, were sensitive to all antimicrobial agents tested. Thus, the molecular typing confirmed that the S. enterica serovar Bovismorbificans outbreak isolates from humans and sprout salad were identical and strongly supported the epidemiological finding that S. enterica serovar Stanley outbreak isolates also originated from contaminated alfalfa seeds. It also confirmed that the sources of similar outbreaks in Sweden in 1994 caused by S. enterica serovar Bovismorbificans and in the United States in 1995 caused by S. enterica serovar Stanley and the source of the Finnish outbreaks were common.
Detection and differentiation of rabbit hemorrhagic disease and European brown hare syndrome viruses by amplification of VP60 genomic sequences from fresh and fixed tissue specimens
Authors: Ros Bascuñana C , Nowotny N , Belák S
Published: January 2020

Two reverse transcription-PCR (RT-PCR) assays have been developed for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), two closely related caliciviruses. In order to select highly specific primers, comparative analysis was performed with a large number of RHDV and EBHSV genomic sequences. Regarding these data, primers were selected from similar regions of the VP60 genes to amplify a fragment of 316 nucleotides from the genome of RHDV and a fragment of 265 nucleotides from the genome of EBHSV. In sensitivity studies, as few as 10 copies of cloned viral genomic fragments were detected in each PCR assay, and no cross amplification was observed between the two viruses. The diagnostic value of the assays was confirmed with clinical material by testing fresh and formalin-fixed, paraffin-embedded liver and spleen specimens from a large number of geographically and temporally distant outbreaks. Thus, the two PCR assays provide highly specific and sensitive, novel means of direct detection of the two caliciviruses. In addition, by detecting the viruses in formalin-fixed, paraffin-embedded tissues (PETs), the RT-PCR assays facilitate retrospective virological and epidemiological studies. For example, the identification of EBHSV in PET specimens collected in the 1970s indicates that this virus appeared in the hare populations several years before the first reports of European brown hare syndrome during the 1980s.
Isolation and characterization of Ehrlichia chaffeensis strains from patients with fatal ehrlichiosis
Authors: Paddock C D , Sumner J W , Shore G M , Bartley D C , Elie R C , McQuade J G , Martin C R , Goldsmith C S , Childs J E
Published: January 2020

Two new isolates of Ehrlichia chaffeensis (designated Jax and St. Vincent) were obtained from patients with fatal ehrlichial infections. Patients developed characteristic manifestations of severe disease due to E. chaffeensis, including marked thrombocytopenia, pulmonary insufficiency, and encephalopathy. Primary isolation was achieved in DH82 cells; the Jax and St. Vincent isolates were detected within 19 and 8 days postinoculation, respectively. The isolates were characterized by molecular evaluation of the 16S rRNA gene, the groESL heat shock operon, a 120-kDa immunodominant protein gene, and an incompletely characterized repetitive-motif sequence (variable-length PCR target [VLPT]). The sequences were compared with those of the corresponding molecular regions in the type isolate (Arkansas). St. Vincent contained one fewer repeat unit in both the 120-kDa protein gene and the VLPT compared with corresponding sequences of the Jax and Arkansas isolates. 16S rRNA gene sequences from the two new isolates had 100% identity to the corresponding sequences of the 91HE17 and Sapulpa isolates of E. chaffeensis, and to the corrected 16S rRNA gene sequence of the Arkansas isolate. The Jax isolate grew more slowly than the St. Vincent isolate in DH82 cells, and both of the new isolates grew more slowly than the extensively passaged Arkansas isolate. Although specific associations between ehrlichial pathogenicity and genotype were not identified from these comparisons, recovery of this organism from a spectrum of clinical presentations remains an integral step in understanding mechanisms of disease caused by E. chaffeensis.
Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness
Authors: Itoh Y , Nagano I , Kunishima M , Ezaki T
Published: January 2020

A massive outbreak of gastrointestinal illness occurred in Tajimi city, Gifu prefecture, in June of 1993 in which 2,697 children in elementary and junior high schools developed severe diarrhea. Stool specimens from 30 children with severe protracted diarrhea were studied. Twenty-seven strains of enteroaggregative Escherichia coli (EAggEC) isolated from 12 of 30 patients all belonged to the same serotype, O untypeable (OUT):H10, and showed the same biochemical characteristics and antibiotic susceptibility pattern. These strains were negative for the virulence factors of the four standard categories of diarrheagenic E. coli (enterotoxigenic, enteropathogenic, enteroinvasive, and enterohemorrhagic). However, the isolates showed an aggregative pattern of adherence to HEp-2 cells and had a 60-MDa plasmid and an astA gene, which encodes heat-stable enterotoxin-1 production. These data suggested that the EAggEC serotype OUT:H10 was associated with this massive outbreak of gastrointestinal illness.
Multiplex AMPLICOR PCR screening for Chlamydia trachomatis and Neisseria gonorrhoeae in women attenting non-sexually transmitted disease clinics. The European Chlamydia Epidemiology Group
Authors: Bassiri M , Mårdh P A , Domeika M
Published: January 2020

A new PCR kit (AMPLICOR CT/NG; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was used as a screening tool for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in first-void urine (FVU) specimens from 3,340 asymptomatic women attending European health care units for contraceptive advice or pregnancy termination. All samples were kept frozen (-20 degrees C) prior to testing. Chlamydia-positive samples were retested once by the plasmid-based PCR kit and also by a major outer membrane protein (MOMP) primer-based PCR. Discrepancies were resolved by using the direct immunofluorescence test (DIF) with the centrifuged sediment of the FVU specimens. Samples positive for N. gonorrhoeae were retested by chromosomal primer-based PCR and verified by a 16S RNA PCR. Of the samples tested, 1.8% were considered inhibitory by using the internal amplification control. Of 81 samples positive for C. trachomatis, 74 samples were positive by both plasmid- and MOMP-based PCRs, 6 samples were positive by plasmid-based PCR and DIF, and one sample was positive by both MOMP-based PCR and DIF. Nine samples (0.3%) were positive for N. gonorrhoeae by the chromosomal primer-based PCR; however, none of the results could be confirmed. The test offers the unique ability to identify inhibition of amplification with the optional internal control.
Molecular epidemiology of a nosocomial outbreak due to SHV-4-producing strains of Citrobacter diversus
Authors: El Harrif-Heraud Z , Arpin C , Benliman S , Quentin C
Published: January 2020

Over a 6-month period, eight strains of Citrobacter diversus (Citrobacter koseri) resistant to extended-spectrum cephalosporins and monobactams were isolated from seven colonized and/or infected patients from the same intensive care unit. All strains harbored a single large conjugative plasmid which mediated an extended-spectrum beta-lactamase of the SHV-4 type (ceftazidimase phenotype; enzyme pI, 7.8; plasmid DNA hybridization with a blaSHV-specific probe). All strains were characterized by antibiotic resistance pattern analysis, beta-lactamase content analysis, plasmid profiling, ribotyping with EcoRI, and arbitrarily primed (AP)-PCR with primers O8 and O12. Among the eight C. diversus strains, strains Cd5 to Cd12, six isolates (isolates Cd6 to Cd11) were identical by all markers; one strain (strain Cd5) differed by two markers (antibiotype and AP-PCR pattern with primer O8), and the remaining strain (strain Cd12) differed by two other markers (ribotype and AP-PCR pattern with primer O12). Our results suggest that six of the eight SHV-4-producing C. diversus strains studied (strains Cd6 to Cd11) were a single epidemic strain. Strain Cd5 could be related to the epidemic strain; the origin of strain Cd12 remains uncertain.
Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis
Authors: Chatellier S , Ramanantsoa C , Harriau P , Rolland K , Rosenau A , Quentin R
Published: January 2020

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).
Failure to detect Chlamydia trachomatis in cell culture by using a monoclonal antibody directed against the major outer membrane protein
Authors: Boman J , Gaydos C , Juto P , Wadell G , Quinn T C
Published: January 2020

Two commercially available monoclonal antibodies for cell culture confirmation of Chlamydia trachomatis were compared in two prospective studies and one large retrospective study. In total, more than 33,000 genital specimens were cultured in parallel and stained with both antibodies, one of which was directed against the major outer membrane protein (MOMP) and one of which was directed against the lipopolysaccharide (LPS). We found the anti-LPS-based assay to be more sensitive and as specific as the anti-MOMP-based assay for C. trachomatis cell culture confirmation of genital specimens.
Recent emergence of new variants of Yersinia pestis in Madagascar
Authors: Guiyoule A , Rasoamanana B , Buchrieser C , Michel P , Chanteau S , Carniel E
Published: January 2020

Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain.
Molecular epidemiology of Ornithobacterium rhinotracheale
Authors: Amonsin A , Wellehan J F , Li L L , Vandamme P , Lindeman C , Edman M , Robinson R A , Kapur V
Published: January 2020

Ornithobacterium rhinotracheale is a recently described gram-negative rod-shaped bacterium associated with respiratory tract infections in poultry. In order to determine the molecular epidemiology of this bacterium, we characterized 55 O. rhinotracheale isolates from eight countries on four continents by multilocus enzyme electrophoresis (MLEE), repetitive sequence based-PCR (rep-PCR), and 16S rRNA gene sequencing. MLEE discriminated the O. rhinotracheale isolates into six electrophoretic types (ETs), of which only three ETs were recovered from domesticated poultry. The 16S rRNA gene sequence and rep-PCR analyses confirmed the results obtained by MLEE and indicated limited heterogeneity among isolates of O. rhinotracheale recovered from poultry. Taken together, the results of our analysis demonstrate that the majority of O. rhinotracheale isolates recovered from domesticated poultry throughout the world are represented by a small group of closely related clones and suggest that the bacterium was recently introduced to domesticated poultry from wild bird populations.