bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of chlorhexidine-silver sulfadiazine-impregnated central venous catheters on in vitro quantitation of catheter-associated bacteria
Authors: Schmitt S K , Knapp C , Hall G S , Longworth D L , McMahon J T , Washington J A
Published: January 2020

To assess the impact of the antiseptic effects of silver sulfadiazine-chlorhexidine-impregnated central venous catheters on catheter culture systems, a series of in vitro experiments was performed. Segments of antiseptic and non-antiseptic-impregnated catheters were sonicated in thioglycolate broth and removed. After the addition of 10(3) CFU of Staphylococcus epidermidis per ml, aliquots of catheter-exposed broth were subcultured onto blood agar at 15-min intervals. Decreased mean colony counts were noted at 45 min for broth exposed to antiseptic-impregnated catheters compared with the colony counts for broth exposed to non-antiseptic-impregnated catheters (170 versus 540 CFU/ml). These effects, which were also demonstrated by the roll-plate method, were abrogated by the use of medium containing inhibitors of silver sulfadiazine and chlorhexidine. To assess the duration of the antiseptic effects, catheter segments were suspended for up to 14 days in phosphate-buffered saline, incubated with 10(6) CFU of S. epidermidis per ml, and cultured. Inhibition of bacterial growth by antiseptic-impregnated catheters disappeared after 14 days. These studies suggest that antiseptic compounds elute from catheters during broth- and solid medium-based culturing processes, making necessary the addition of inhibitors of these compounds in culture media. They further suggest that the antimicrobial effects of antiseptic-impregnated catheters wane within several days of placement.
Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture
Authors: Karch H , Janetzki-Mittmann C , Aleksic S , Datz M
Published: January 2020

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.
Molecular epidemiology of Klebsiella pneumoniae producing SHV-5 beta- lactamase: parallel outbreaks due to multiple plasmid transfer
Authors: Prodinger W M , Fille M , Bauernfeind A , Stemplinger I , Amann S , Pfausler B , Lass-Florl C , Dierich M P
Published: January 2020

Over a period of 22 months, 32 patients treated in three independent intensive care units of the Innsbruck University Hospital were infected with extended-spectrum beta-lactamase-producing members of the family Enterobacteriaceae (30 Klebsiella pneumoniae isolates, 1 Klebsiella oxytoca isolate, and 1 Escherichia coli isolate). As confirmed by sequencing of a bla gene PCR fragment, all isolates expressed the SHV-5-type beta-lactamase. Genomic fingerprinting of epidemic strains with XbaI and pulsed-field gel electrophoresis grouped 20 of 21 isolates from ward A into two consecutive clusters which included 1 of 3 ward B isolates. All six K. pneumoniae isolates from ward C formed a third cluster. Stool isolates of asymptomatic patients and environmental isolates belonged to these clusters as well. Additionally, 2,600 routine K. pneumoniae isolates from the surrounding provinces (population, 900,000) were screened for SHV-5 production. Only one of six nonepidemic isolates producing SHV-5 beta-lactamase was matched with the outbreak strains by genomic fingerprinting. Plasmid fingerprinting, however, revealed the epidemic spread of a predominant R-plasmid, with a size of approximately 80 kb, associated with 29 of the 30 K. pneumoniae isolates. This plasmid was also present in the single K. oxytoca and E. coli isolates from ward C and in three nonepidemic isolates producing SHV-5. Our results underline that strain typing exclusively on the genomic level can be misleading in the epidemiological investigation of plasmid-encoded extended-spectrum beta-lactamases. Our evidence for multiple events of R-plasmid transfer between species of the family Enterobacteriaceae in this nosocomial outbreak stresses the need for plasmid typing, especially because SHV-5 beta-lactamase seems to be regionally spread predominantly via plasmid transfer.
Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis
Authors: Hla S W , Hui K P , Tan W C , Ho B
Published: January 2020

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.
Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis
Authors: Guttman D S , Wang P W , Wang I N , Bosler E M , Luft B J , Dykhuizen D E
Published: January 2020

The genetic heterogeneity of the spirochete Borrelia burgdorferi within single adult black-legged ticks from Shelter Island, N.Y., was determined by cold, single-strand conformation polymorphism (SSCP) analysis. The central region of the ospA gene of B. burgdorferi from infected ticks was amplified by nested PCR. Amplified product of the correct size was obtained from 20 to 45 ticks (44%). This is the fraction of ticks that is expected to be infected with B. burgdorferi. Four variant classes were determined by SSCP analysis. Eight ticks were infected with a single variant, nine ticks were infected with two variants, two ticks were infected with three variants, and one tick was infected with all four variants. DNA from each variant was sequenced. Five different sequences were found. The sequence of each variant was different from that of another variant by a single base. SSCP analysis could distinguish three of the four single-base changes found in the region.
U.S. hospital mycobacteriology laboratories: status and comparison with state public health department laboratories
Authors: Tokars J I , Rudnick J R , Kroc K , Manangan L , Pugliese G , Huebner R E , Chan J , Jarvis W R
Published: January 2020

In response to the resurgence of tuberculosis, the Centers for Disease Control and Prevention recommended the use of certain mycobacteriology laboratory methods to improve the accuracy of diagnosis and/or minimize times to complete specimen processing. A study to determine the extent to which these recommended methods were being used in hospital laboratories was needed. In 1992, a survey was mailed to infection control and laboratory personnel at 1,076 hospitals with > or = 100 beds to determine the mycobacterial laboratory services being performed, the methods being used, the number of specimens being processed, and the times to completion during 1991. In 1995, a 20% sample of hospital laboratories that responded to the initial questionnaire was resurveyed. Responses to the 1992 survey were received from personnel at 756 (70%) hospitals representing 750 laboratories. Among laboratories performing the services, the use of recommended methods was as follows: fluorochrome stain for acid-fast bacillus microscopy (47%); radiometric methods for primary culture (29%); rapid (radiometric methods, use of nucleic acid probes, high-performance liquid chromatography, or gas-liquid chromatography) methods for identification of Mycobacterium tuberculosis (59%); and radiometric methods for drug susceptibility testing (55%). Reported times to complete specimen processing were shortest for laboratories that used recommended methods and longest for hospitals that referred specimens to outside laboratories. Only 46% of surveyed laboratories performed at least the minimal number of mycobacterial cultures (20/week) deemed necessary to maintain competence. Among 145 laboratories that performed the services and were resurveyed in 1995, use of recommended techniques increased from 44 to 73% for acid-fast bacillus microscopy, from 27 to 37% for primary culture, from 59 to 88% for M. tuberculosis identification, and from 55 to 75% for drug susceptibility testing. These changes were associated with reductions in reported specimen turnaround times. Use of the methods recommended by the Centers for Disease Control and Prevention increased at the resurveyed hospital mycobacteriology laboratories between 1991 and 1995. However, continued efforts are needed to increase the use of recommended methods at moderate- and high-volume laboratories, encourage referral of specimens from low-volume laboratories, and transmit results rapidly from all laboratories.
Controlled study of cytolethal distending toxin-producing Escherichia coli infections in Bangladeshi children
Authors: Albert M J , Faruque S M , Faruque A S , Bettelheim K A , Neogi P K , Bhuiyan N A , Kaper J B
Published: January 2020

The role of cytolethal distending toxin (CDT)-producing Escherichia coli, a newly described category of E. coli, in the causation of diarrhea was studied by screening E. coli isolates from 546 children < 5 years of age with diarrhea and 215 matched controls without diarrhea by using a specific DNA probe. Although CDT-positive E. coli strains were isolated from more children with diarrhea than from healthy controls (3.1 versus 0.93%), this difference did not reach statistical significance (P = 0.082). All CDT-positive strains also possessed the virulence factors of enteropathogenic E. coli or enteroaggregative E. coli isolates.
Isolation of group B porcine rotavirus in cell culture
Authors: Sanekata T , Kuwamoto Y , Akamatsu S , Sakon N , Oseto M , Taniguchi K , Nakata S , Estes M K
Published: January 2020

While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured. In this study we successfully isolated porcine group B rotavirus in swine kidney cells. Pancreatin treatment is essential for the propagation of group B rotavirus.
Serotyping and esterase typing for analysis of Listeria monocytogenes populations recovered from foodstuffs and from human patients with listeriosis in Belgium
Authors: Gilot P , Genicot A , André P
Published: January 2020

Listeria monocytogenes strains isolated in Belgium from different foodstuffs and in sporadic cases of human listeriosis were analyzed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human patients with listeriosis were further studied by esterase typing. The twenty esterase patterns defined were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C and the pathogenicity level of strains in immunocompromised mice could not explain the unequal distribution of esterase types. The discrimination index of esterase typing (DI = 0.868) was compared with that of serotyping (DI = 0.666) and with that of the two combined methods (DI = 0.899).
Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever
Authors: Thong K L , Passey M , Clegg A , Combs B G , Yassin R M , Pang T
Published: January 2020

Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.
Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex
Authors: Liébana E , Aranaz A , Francis B , Cousins D
Published: January 2020

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.
Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin
Authors: Didier E S , Rogers L B , Brush A D , Wong S , Traina-Dorge V , Bertucci D
Published: January 2020

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.
Gender is not a factor in serum human immunodeficiency virus type 1 RNA levels in patients with viremia
Authors: Bush C E , Donovan R M , Markowitz N , Baxa D , Kvale P , Saravolatz L D
Published: January 2020

We investigated gender as a factor in viral load measurements for human immunodeficiency virus-infected patients. Forty antiretroviral-therapy-naive, age- and CD4-matched women and men were tested for serum RNA and p24 antigen levels prior to antiretroviral therapy and at approximately 12 weeks after therapy. No gender differences were observed for these two markers of viral load.
Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA
Authors: da Silva A J , Schwartz D A , Visvesvara G S , de Moura H , Slemenda S B , Pieniazek N J
Published: January 2020

Enterocytozoon bieneusi is the most common microsporidian infecting patients with AIDS. We have developed a PCR primer pair, named EBIEF1/EBIER1, based on the small-subunit rRNA sequence of this microsporidian. Compared with other PCR-based methods, this primer pair shows a higher efficiency of detection in diagnostic applications than does another previously described primer pair, V1/EB450.
Vertebral osteomyelitis due to Staphylococcus lugdunensis
Authors: Murdoch D R , Everts R J , Chambers S T , Cowan I A
Published: January 2020

We present the first reported case of vertebral osteomyelitis due to Staphylococcus lugdunensis. The infection occurred in an 80-year-old woman who had been taking glucocorticosteroids. S. lugdunensis is a coagulase-negative staphylococcus with considerable potential as a human pathogen. Isolation of this organism should be regarded as significant unless evidence suggests otherwise.
Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR
Authors: Busch U , Hizo-Teufel C , Boehmer R , Fingerle V , Nitschko H , Wilske B , Preac-Mursic V
Published: January 2020

A total of 36 European Borrelia burgdorferi sensu lato cerebrospinal fluid isolates (mainly from southern Germany) were analyzed by pulsed-field gel electrophoresis (PFGE) for large restriction fragment pattern (LRFP) and linear plasmid profiles. Analyzing this large panel of isolates, we detected all three species of B. burgdorferi sensu lato pathogenic for humans in cerebrospinal fluid from patients with Lyme neuroborreliosis by PFGE typing after MluI digestion: 21 B. garinii (58%), 10 B. afzelii (28%), and 4 B. burgdorferi sensu stricto (11%) strains as well as 1 isolate with bands characteristic of both B. afzelii and B. garinii. Species classification by PFGE typing was confirmed by 16S rRNA-specific PCR. Eighteen isolates (11 B. garinii, 6 B. afzelii, and 1 B. burgdorferi sensu stricto isolate) were further characterized by LRFP with four different restriction enzymes (ApaI, KspI, SmaI, and XhoI). All B. afzelii isolates showed identical patterns for each restriction enzyme group. Considerable heterogeneity was demonstrated within the B. garinii group. Subsequent analysis of plasmid profiles revealed only marginal differences for B. afzelii strains but different patterns for B. garinii isolates. In one B. afzelii strain we found a linear plasmid of about 110 kbp not described before. LRFP analysis by PFGE is a suitable tool for the molecular characterization of B. burgdorferi sensu lato strains and allows determination not only of the species but also of the subtypes within B. garinii.
Detecting bunyaviruses of the Bunyamwera and California serogroups by a PCR technique
Authors: Kuno G , Mitchell C J , Chang G J , Smith G C
Published: January 2020

Many bunyaviruses of the Bunyamwera and California serogroups are medically important human pathogens. The development of an effective technique to detect the viruses by using molecular biologic tools, such as PCR, improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, we evaluated eight pairs of primers for reactivity with 44 viruses of the genus Bunyavirus, using a reverse transcriptase PCR technique. With a pair of serogroup-specific primers we designed, all viruses of the serogroups tested could be detected. Further, virus-specific primer pairs were identified for California encephalitis virus, Jamestown Canyon virus, La Crosse virus, and snowshoe hare virus for use in North America. Using this technique, we could detect one La Crosse virus-infected mosquito in a pool of 100 mosquitoes with undetectable plaque titers.
Antimicrobial susceptibilities of Lactococcus lactis and Lactococcus garvieae and a proposed method to discriminate between them
Authors: Elliott J A , Facklam R R
Published: January 2020

The MICs of antimicrobial agents contained in the SCEPTOR Streptococcus MIC panels (Becton Dickinson Microbiology Systems) were determined for Lactococcus lactis, L. garvieae, and unknown Lactococcus species. Several isolates had reduced susceptibilities to many of the antimicrobial agents contained in the panel. For L. garvieae, the MICs of penicillin and, possibly, cephalothin were higher than for L. lactis, and unlike L. lactis, L. garvieae was resistant to clindamycin, indicating that knowledge of the Lactococcus species causing an infection might influence the choice of antimicrobial therapy. Susceptibility to clindamycin can also be used to differentiate between L. lactis and L. garvieae.
Detection of Borrelia burgdorferi-specific DNA in urine specimens from patients with erythema migrans before and after antibiotic therapy
Authors: Schmidt B , Muellegger R R , Stockenhuber C , Soyer H P , Hoedl S , Luger A , Kerl H
Published: January 2020

A nested PCR was developed for the detection of Borrelia burgdorferi-specific DNA in the urine of patients with erythema migrans. The target for the nested PCR was a specific region of the flagellin gene; the detection limit was less than five organisms of B. burgdorferi including all three species B. burgdorferi sensu stricto, B. afzelii, and B. garinii. A prospective, controlled, blinded study was performed with 26 patients with erythema migrans to evaluate the nested PCR method with clinical samples. B. burgdorferi-specific DNA could be detected in urine specimens from 22 of 24 patients with erythema migrans (sensitivity, 91.61%). Immediately after therapy, 11 of 19 patients still yielded positive results (58%). Eight weeks after therapy, 2 of 16 patients (13%) were positive by PCR of urine, and 20 weeks after treatment none of seven investigated urine samples was reactive. Essential for the sensitivity that was obtained was the development of a simple DNA extraction procedure. The results of the study indicate that the described method is highly sensitive and allows for the effective control of the efficacy of antibiotic therapy in patients with early Lyme borreliosis.
Rapid susceptibility testing for nontuberculosis mycobacteria using flow cytometry
Authors: Bownds S E , Kurzynski T A , Norden M A , Dufek J L , Schell R F
Published: January 2020

We demonstrated previously that susceptibility testing of Mycobacterium tuberculosis could be accomplished within 24 h after the organisms were incubated with antituberculosis agents by using fluorescein diacetate (FDA) staining and flow cytometry. Continued studies have now shown that assay suspensions containing M. avium, M. fortuitum, M. gordonae, or M. marinum incubated with various concentrations of ciprofloxacin, clarithromycin, erythromycin, kanamycin, rifampin, tobramycin hydrolyzed less FDA than drug-free controls. Suspensions of treated and nontreated mycobacteria could be easily differentiated at 6 and 24 h after the initiation of the susceptibility assays by using FDA staining and flow cytometry. In addition, multiplication of the mycobacteria was not required to discern differences between drug-free suspensions of mycobacteria and those treated with antimycobacterial agents. The flow cytometric assay is simple, reproducible, and rapid.
Isolation of Ehrlichia canis from dogs following subcutaneous inoculation
Authors: Gaunt S D , Corstvet R E , Berry C M , Brennan B
Published: January 2020

Subcutaneous inoculation of dogs with Ehrlichia canis was investigated as a more appropriate model of canine ehrlichiosis, which is naturally transmitted by arthropod vectors. A dose-dependent response occurred following subcutaneous inoculation of seven groups of dogs with log concentrations of E. canis-infected canine-origin cells. Ehrlichial infection in dogs was defined as concurrence of an increased titer of anti-E. canis immunoglobulin G (IgG) antibody in serum, a decreased platelet concentration, and isolation of E. canis by blood culture. In dogs administered the two lowest doses, no changes were detected. In seven of nine dogs administered three intermediate doses, the only change detected was a transient and mild increase in the anti-E. canis IgG antibody titer in serum. Only two of nine dogs inoculated with the intermediate doses developed an ehrlichial infection. Five of six dogs administered the two highest dose of E. canis developed an ehrlichial infection. These dogs had the highest IgG antibody titers in serum and the earliest isolation of E. canis from blood. In dogs that developed an ehrlichial infection, thrombocytopenia occurred by 28 days after inoculation, while increased IgG antibody titers in serum and blood cultures positive for E. canis occurred as early as 14 days postinoculation. Thrombocytopenia and seroconversion occurred later in the course of infection than previously reported for ehrlichial infections induced by intravenous inoculation. The route of administration and E. canis inoculum size can influence the course of ehrlichial infection and should be regarded as important variables in experimentally induced canine ehrlichiosis.
Detection of quinolone-resistant Neisseria gonorrhoeae
Authors: Kam K M , Wong P W , Cheung M M , Ho N K
Published: January 2020

The present National Committee for Clinical Laboratory Standards (NCCLS) guideline for testing Neisseria gonorrhoeae quinolone susceptibility defines only a susceptible category for ciprofloxacin, enoxacin, lomefloxacin, and ofloxacin, while susceptible, intermediate, and resistant categories are defined for fleroxacin. To further define the criteria for detection of quinolone resistance in gonococci, by standard disk diffusion and agar dilution methodologies recommended by the NCCLS, we tested 29 strains of quinolone-resistant N. gonorrhoeae (QRNG) recently isolated from ofloxacin-treated patients who were considered clinical failures. Regression analyses were performed on these results together with those of another 20 strains showing reduced susceptibility and 13 fully susceptible strains (ofloxacin MICs of < or = 0.25 microgram/ml). With 5-micrograms ofloxacin disks, resistance in 27 (93.1%) of the QRNG strains (MICs of > 1 microgram/ml) was detected by the criterion of a zone diameter of < 22 mm, while in the remaining 2 (6.9%), the disks failed to detect resistance. A cluster of 15 highly resistant strains showed ofloxacin MICs of > 4 micrograms/ml and zone diameters of < 13 mm. When tested with 5-micrograms ciprofloxacin disks, the corresponding values for resistance and high-level resistance of these QRNG strains were < 25 mm (MICs of > 0.5 micrograms/ml) and < 15 mm (MICs of > 2 micrograms /ml), respectively. Six strains for which ofloxacin MICs were > or = 8 micrograms/ml showed no zones at all with both 5-micrograms ofloxacin and 5-micrograms ciprofloxacin disks. These QRNG strains are now firmly established in the Southeast Asia region, and it is important for clinical laboratories to recognize these clinically resistant strains and to monitor their spread.
Detection of enteroviruses from clinical specimens by spin amplification shell vial culture and monoclonal antibody assay
Authors: Klespies S L , Cebula D E , Kelley C L , Galehouse D , Maurer C C
Published: January 2020

Conventional tube cell culture was compared with a 72-h, spin-amplified shell vial indirect immunofluorescence assay for the detection of enterovirus from clinical specimens. The sensitivity for the shell vial assay after resolution of discrepant results were 93 and 100%, respectively. The shell vial assay detected 93% of the positive cultures within 72 h of incubation while conventional tube culture detected only 51% of the positive cultures within the same time interval. The data suggest that a spin-amplified shell vial indirect immunofluorescence assay may be useful for the detection of enterovirus from clinical specimens.
Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein
Authors: Dea S , Gagnon C A , Mardassi H , Milane G
Published: January 2020

Two hybridoma cell lines producing monoclonal antibodies (Mabs) to the 19-kDa matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from BALB/c mice that were immunized with a reference Quebec tissue culture-adapted strain (strain IAF-Klop). The polypeptide specificities of the MAbs were determined by immunoblotting and immunoprecipitation tests with concentrated and purified preparations of the virus and by determining their reactivities with the Escherichia coli-expressed gene products of open reading frames 5 to 7. The two anti-M protein MAbs (MAbs IAFK3 and IAFK6) and another MAb (MAb IAFK8) directed to the 15-kDa nucleocapsid (N) protein were devoid of virus-neutralizing activity. A library of four anti-N protein MAbs (MAbs IAFK8, SDOW17, VO17, and EP147) and two anti-M protein MAbs (MAbs IAFK6 and IAFK3) was used to investigate, by an indirect fluorescent-antibody assay, the antigenic diversity of 15 Canadian PRRSV isolates, in comparison with those of the U.S. ATCC VR2332 attenuated vaccine strain and two reference European (Lelystad and Weybridge) PRRSV strains. The North American and European PRRSV isolates tested shared the epitopes recognized by anti-N protein MAbs IAFK8 and SDOW17, but three distinct patterns could be identified on the basis of their reactivities with the other anti-PRRSV MAbs. No reactivity to the anti-M protein MAbs was observed by either European PRRSV isolate or the attenuated U.S. vaccine strain.
Karyotype instability in Cryptococcus neoformans infection
Authors: Fries B C , Chen F , Currie B P , Casadevall A
Published: January 2020

The electrophoretic karyotypes of 32 clinical and 3 environmental Cryptococcus neoformans isolates from New York City were studied by contour clamped homogeneous electrophoresis. There was extensive variation among the electrophoretic karyotypes of isolates from different patients. Sequential C. neoformans isolates from patients with chronic or relapsing infection had very similar karyotypes. However, minor differences in electrophoretic karyotypes were detected among sequential isolates from 50% of the patients studied, suggesting the occurrence of chromosomal rearrangements or deletions in vivo. This hypothesis was tested by infecting mice, recovering isolates from mouse organs, and comparing the electrophoretic karyotypes before and after passage. Three clinical and three environmental strains were studied before and after passage in mice. Karyotype differences were detected after mouse passage for one clinical and two environmental strains. Our results indicate (i) extensive karyotype variation among isolates from a small geographic regions, (ii) a high frequency of electrophoretic karyotype differences among sequential isolates from individual patients, and (iii) the occurrence of electrophoretic karyotype changes during experimental infection of mice. The implications of these observations are discussed.
Genetic dissimilarity of two fluconazole-resistant Candida albicans strains causing meningitis and oral candidiasis in the same AIDS patient
Authors: Berenguer J , Diaz-Guerra T M , Ruiz-Diez B , Bernaldo de Quiros J C , Rodriguez-Tudela J L , Martinez-Suarez J V
Published: January 2020

We describe a patient with AIDS who simultaneously developed Candida meningitis with three positive cerebrospinal fluid cultures and oral candidiasis. This patient also had a history or recurrent episodes of oral candidiasis treated with fluconazole. The patient did not respond to this therapy but was cured with amphotericin B and flucytosine. In vitro susceptibility tests revealed that each infection was caused by fluconazole-resistant Candida albicans isolates. Strain delineation by karyotyping, NotI restriction pattern analysis, hybridization with the specific probe 27A, and PCR fingerprinting with the phage M13 core sequence clearly demonstrated that meningitis and oral thrush were caused by two genetically different isolates.
Genetic characterization of rabies field isolates from Venezuela
Authors: de Mattos C A , de Mattos C C , Smith J S , Miller E T , Papo S , Utrera A , Osburn B I
Published: January 2020

Twenty samples from cases of rabies in humans and domestic animals diagnosed in Venezuela between 1990 and 1994 and one sample from a vampire bat collected in 1976 were characterized by reactivity to monoclonal antibodies against the viral nucleoprotein and by patterns of nucleotide substitution in the nucleoprotein gene. Three antigenic variants were found: 1, 3, and 5. Antigenic variant 1 included all samples from dogs and humans infected by contact with rabid dogs. Unique substitutions permitted identification of two separate outbreaks of dog rabies in the Maracaibo Depression and Los Llanos region and in the Andean region of Venezuela. Samples from the vampire bat and two head of cattle were characterized as antigenic variant 3 and showed a nucleotide sequence homology of 96 to 98% to each other and to samples of vampire bat-associated rabies throughout Latin America. Ten of the remaining 12 samples were characterized as antigenic variant 5. Genetic studies indicated that 11 of these samples formed a highly homologous and distinctive group but were closely related to samples of vampire bat-associated rabies. The 12th sample of variant 5 (from a cat) showed only 78 to 80% genetic homology to samples of rabies associated with vampire bats. The application of antigenic and genetic typing to rabies surveillance in Latin America is essential to improve control programs. Recognition of the source of outbreaks of dog rabies and identification of wildlife species maintaining sylvatic cycles of rabies transmission permit better utilization of public health resources.
Evaluation of nutritive capacities of 10 broth media
Authors: Scythes K D , Louie M , Simor A E
Published: January 2020

Ten broth media were evaluated for their ability to support the growth of low inocula of selected fastidious aerobic, microaerophilic, and anaerobic bacteria. Thioglycolate medium USP (Oxoid, Basingstoke, England), cooked meat broth (Oxoid), and fastidious anaerobe broth (Quelab Laboratories Inc., Montreal, Quebec, Canada) were best able to support the growth of the greatest variety of organisms at the lowest inoculum tested (10(1) CFU). In most cases, incubation of these broths resulted in visible turbidity within 3 days. These three broth media were found to be equally suitable for use as an enrichment broth in the clinical laboratory.
Oerskovia xanthineolytica infection of a prosthetic joint: case report and review
Authors: Harrington R D , Lewis C G , Aslanzadeh J , Stelmach P , Woolfrey A E
Published: January 2020

Oerskovia spp. are gram-positive, Nocardia-like bacilli which inhabit the soil and rarely cause human infections. Previously reported cases of Oerskovia infection have been characterized by a nonaggressive course and an association with foreign bodies. We report the first case of a patient with a prosthetic joint infection due to Oerskovia xanthineolytica. Our patient presented with a prolonged, indolent course and was thought to have aseptic loosening of his prosthesis until the time of surgery. He was cured of his infection by removal of the prosthesis, antibiotic therapy, and delayed reimplantation. Review of the previous 10 reported cases of Oerskovia infection in humans supports the recommendation that foreign-body-associated infections should be treated with a strategy that includes removal of the foreign material.
Phage specific for Vibrio cholerae O139 Bengal
Authors: Albert M J , Bhuiyan N A , Rahman A , Ghosh A N , Hultenby K , Weintraub A , Nahar S , Kibriya A K , Ansaruzzaman M , Shimada T
Published: January 2020

From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V. cholerae O139 strains only was isolated. The phage is useful for the confirmatory diagnosis of V. cholerae O139 infection and for the differentiation of variants that lack the capsule.
Clinical and microbiological characteristics of Flavobacterium indologenes infections associated with indwelling devices
Authors: Hsueh P R , Teng L J , Ho S W , Hsieh W C , Luh K T
Published: January 2020

Clinical infections caused by Flavobacterium indologenes have never been documented. Thirteen isolates derived from seven patients with indwelling device-associated F. indologenes infections were identified from 1 April through 30 November 1995. The antimicrobial susceptibilities to 20 antimicrobial agents of the isolates, the cellular fatty acid chromatograms for the isolates, and the random amplified polymorphic DNA (RAPD) patterns generated by arbitrarily primed PCR of the isolates were studied. The antibiotypes and RAPD patterns differed among the isolates recovered from different patients. However, both antibiotypes and RAPD patterns were identical among the five isolates from one patient with multiple episodes of central venous catheter-associated bacteremia within a 1.5-month period and between the two isolates from another patient suffering from two episodes of catheter-related bacteriuria at an interval of 14 days. It is documented that the recurrent infections in each of these two patients were caused by a single F. indologenes clone, respectively. Identical antibiotypes and RAPD patterns were also demonstrated between two isolates from a patient with ventilator-associated pneumonia, one recovered from an endotracheal aspirate and the other derived from a blood specimen 10 days later, indicating the invasive nature of F. indologenes. Two cellular fatty acid chromatograms were identified among these isolates. All of the isolates showed in vitro resistance to cephalothin, cefotaxime, ceftriaxone, moxalactam, aztreonam, aminoglycosides, erythromycin, clindamycin, vancomycin, and teicoplanin. F. indologenes should be included as an etiologic agent of infections associated with the use of indwelling devices.
Clinical characterization of a competitive PCR assay for quantitative testing of hepatitis C virus
Authors: Miskovsky E P , Carrella A V , Gutekunst K , Sun C A , Quinn T C , Thomas D L
Published: January 2020

Rational clinical application of quantitative assessments of hepatitis C virus (HCV) RNA depends on an understanding of factors affecting the assay and its intrinsic variability. The effects of three types of blood collection tubes, two storage temperatures, five processing times, and two laboratories on a commercially available quantitative reverse transcriptase PCR assay (AMPLICOR HCV MONITOR) were evaluated. HCV RNA concentrations were assessed in 356 specimens representing 178 aliquots from nine patients. In a multivariate generalized linear model, HCV RNA concentrations decreased when centrifugation was delayed more than 6 h (P = 0.005) and were marginally different between laboratories (P = 0.06), but precentrifugation storage temperature (P = 1.00) and anticoagulation (P = 0.22) had no effect. After adjusting for other factors, the HCV concentration of 95% of a subject's samples were within 0.44 log. Specimens procured for reverse transcriptase PCR-based quantitative HCV testing should be centrifuged within 6 h of collection. Serial assessments should ideally be performed in the same laboratory, and changes in HCV RNA concentration of less than 0.44 log may not be biologically important.
Efficacy of immunoglobulin M serodiagnostic test for rapid diagnosis of acute babesiosis
Authors: Krause P J , Ryan R , Telford S , Persing D , Spielman A
Published: January 2020

To evaluate the efficacy of an immunoglobulin M indirect immunofluorescent-antibody procedure for diagnosing acute babesiosis, we tested patients with acute babesiosis from a site in New England where the disease is enzootic. The sensitivity of the test was 91%, the specificity was 99%, the positive predictive value was 86%, and the negative predictive value was 99%. This B. microti immunoglobulin M indirect immunofluorescent-antibody procedure is sufficiently sensitive, specific, and reproducible for use in the routine clinical diagnosis of acute babesiosis.
Immunoglobulin M immunoblot for diagnosis of Borrelia burgdorferi infection in patients with acute facial palsy
Authors: Jain V K , Hilton E , Maytal J , Dorante G , Ilowite N T , Sood S K
Published: January 2020

We used immunoblotting to improve the specificity of the serologic diagnosis of Lyme borreliosis in cases of acute facial palsy. Twelve of 15 patients (80%) with suspected Lyme borreliosis, versus 0 of 10 controls, were positive by immunoglobulin M immunoblotting of acute-phase sera and 3 were negative, including 2 with borderline enzyme immunoassay results. Immunoglobulin M immunoblotting is a useful test to confirm Borrelia burgdorferi infection in patients with acute facial palsy and a positive enzyme immunoassay result.
Monitoring of cytomegalovirus infection and disease in bone marrow recipients by reverse transcription-PCR and comparison with PCR and blood and urine cultures
Authors: Gozlan J , Laporte J P , Lesage S , Labopin M , Najman A , Gorin N C , Petit J C
Published: January 2020

Preemptive therapy is a promising strategy for the prevention of serious cytomegalovirus (CMV) disease after bone marrow (BM) transplantation but requires relevant diagnostic tests. We compared the clinical value of a reverse transcription (RT)-PCR method, which detected a late viral mRNA in peripheral blood leukocytes (PBL), with a PCR method that detected the viral DNA in PBL and with viral culture from leukocytes and urine for the diagnosis of symptomatic CMV infection after BM transplantation. Forty-five consecutive BM recipients were prospectively tested at weekly intervals by the four methods. CMV infection, demonstrated either by the culture of CMV or by repeated detection of viral DNA, was observed in 28 patients, but only 14 developed CMV-related clinical symptoms. The clinical sensitivity and specificity of each technique for detection of symptomatic infection were, respectively, 36 and 74% for urine culture, 43 and 84% for leukocyte culture, 100 and 65% for PCR, and 71 and 94% for RT-PCR. Although PCR detection of DNA in PBL was the earliest and most sensitive technique for the diagnosis of CMV infection, RT-PCR was more predictive of the onset of CMV-related clinical symptoms. These data suggest that both molecular methods should be used for identifying BM recipients at highest risk of CMV disease.