bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific recognition of purified Cu,Zn superoxide dismutase from Aspergillus fumigatus by immune human sera
Authors: Hamilton A J , Holdom M D , Hay R J
Published: January 2020

Sera from 22 (84.6%) of 26 patients with confirmed Aspergillus infections recognized a purified Cu,Zn superoxide dismutase from A. fumigatus on Western blots (immunoblots). Sera from 32 (71%) of 45 patients with suspected Aspergillus infections were reactive to the superoxide dismutase. Normal human sera and sera from patients with other infections were unreactive.
Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa
Authors: Grundmann H , Schneider C , Hartung D , Daschner F D , Pitt T L
Published: January 2020

We assessed the capacity of three DNA typing techniques to discriminate between 81 geographically, temporally, and epidemiologically unrelated strains of Pseudomonas aeruginosa. The methods, representing powerful tools for hospital molecular epidemiology, included hybridization of restricted chromosomal DNA with toxA and genes coding for rRNA (rDNA) used as probes and macrorestriction analysis of SpeI-digested DNA by pulsed-field gel electrophoresis. The probe typing techniques were able to classify all strains into a limited number of types, and the discriminatory powers were 97.7 and 95.6% for toxA and rDNA typing, respectively. Strains that were indistinguishable on the basis of both toxA and rDNA types defined 12 probe type homology groups. Of these, one contained five strains, three contained three strains each, and eight groups were represented by two strains each. Strains in 10 of the homology groups had the same O serotype. SpeI macrorestriction patterns discriminated between all strains with at least four band differences, which corresponded to a similarity level of 85%. Fifteen pairs of strains were similar at a level of > 75% and differed by only four to seven bands. Of these pairs, 11 belonged to the same probe type homology group, indicating their clonal relatedness. We conclude that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains. However, DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.
Accuracy and interlaboratory reliability of human papillomavirus DNA testing by hybrid capture
Authors: Schiffman M H , Kiviat N B , Burk R D , Shah K V , Daniel R W , Lewis R , Kuypers J , Manos M M , Scott D R , Sherman M E
Published: January 2020

Epidemiologists and clinicians wishing to introduce human papillomavirus (HPV) testing into cervical cancer prevention programs need standardized, reliable, and accurate HPV DNA tests that can detect the full spectrum of pathogenic HPV types. The Hybrid Capture System assay from Digene (hybrid capture assay) is a nonradioactive kit designed to detect 14 HPV types in two groups: a mix of 9 high-risk types associated with anogenital cancer (HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56) and another group of 5 low-risk types associated with condyloma acuminatum (HPV types 6, 11, 42, 43, and 44). The assay yields quantitative data meant to reflect viral concentration. In a study of 199 cervical specimens from women with concurrent Pap smears, we assessed the reliability of the new assay by comparing the hybrid capture assay results from three laboratories. We assessed the accuracy of the hybrid capture assay in comparison with a reference standard of HPV DNA content (multiple testing by several methods in two reference laboratories). We also compared the hybrid capture assay results with the concurrent cytologic diagnoses on the basis of an independent review of each smear by five pathologists. Pairwise interlaboratory agreement rates on HPV positivity for either high-risk or low-risk types ranged from 87 to 94%, and kappa values ranged from 0.61 to 0.83. Among specimens positive for high-risk types (the most important clinical outcome), the interlaboratory correlations of the quantitative data ranged from 0.60 to 0.90. Test results from all three laboratories were strongly associated with those of the HPV DNA reference standard and with the concurrent cytopathologic diagnoses. The most common errors were sporadic, apparently false-positive results.
Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients
Authors: Demaerschalck I , Ben Messaoud A , De Kesel M , Hoyois B , Lobet Y , Hoet P , Bigaignon G , Bollen A , Godfroid E
Published: January 2020

Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.
Study on reliability of commercially available hepatitis C virus antibody tests
Authors: Feucht H H , Zöllner B , Polywka S , Laufs R
Published: January 2020

The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany. The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli. The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA). Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined. In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests. In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR. In follow-up studies conducted over 1 year, these results did not change. In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative. All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later. In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0. In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR. All three patients were free of complaints. The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%). This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.
Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning
Authors: Ahmed R , Sankar-Mistry P , Jackson S , Ackermann H W , Kasatiya S S
Published: January 2020

Bacillus cereus is responsible for an increasing number of food poisoning cases. By using 12 bacteriophages isolated from sewage, a typing scheme for B. cereus isolates from outbreaks or sporadic cases of food poisoning was developed. The phages belonged to three morphotypes. Ten phages with contractile tails and icosahedral heads were members of the Myoviridae family, and two phages with noncontractile tails belonged to the Siphoviridae family. Phage 11 represented a new species. It had an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known. The vast majority of 166 B. cereus strains (161, or 97%) isolated from food poisoning cases were typeable. Of 146 strains isolated from 18 outbreaks, 142 (97%) could be divided into 17 phage types. A good correlation, on the order of 80 to 100%, between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed. Most Bacillus thuringiensis strains were also typeable, providing further evidence of the close relatedness of B. cereus and B. thuringiensis. This phage typing scheme can be a valuable epidemiological tool in tracing the origins of food poisoning caused by B. cereus.
Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay
Authors: Jahn B , Martin E , Stueben A , Bhakdi S
Published: January 2020

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.
Specific bovine brucellosis diagnosis based on in vitro antigen-specific gamma interferon production
Authors: Weynants V , Godfroid J , Limbourg B , Saegerman C , Letesson J J
Published: January 2020

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of cytoplasmic proteins from Brucella melitensis B115 was used as a specific antigenic stimulus in bovine whole blood culture. Supernatants harvested at 18 to 24 h after the in vitro antigenic stimulus were assayed for their gamma interferon (IFN-gamma) content by using a commercial sandwich enzyme-linked immunosorbent assay kit. The IFN-gamma assay was evaluated with 10 heifers during the course (80 days) of an experimental infection and with 14 cows from an ongoing brucellosis outbreak. All of these animals were slaughtered, and pertinent organs were subjected to classical bacteriological analyses. In addition, we analyzed 23 field cases in which false-positive serological reactions occurred. The IFN-gamma results were compared with those of the standard DTH and a battery of serological assays, and they were correlated with bacteriological data. Both for the experimental infection and for the field brucellosis outbreak, the IFN-gamma assay detected infection in more animals than any combination of the serological tests, and it detected infection earlier than these tests. Finally, none of the samples from cows showing false-positive serological reactions was classified as positive by the IFN-gamma assay, attesting to its specificity and to its usefulness in interpreting ambiguous serological results. A rapid and convenient alternative to the DTH, the IFN-gamma assay appears to be an ideal method that is complementary to the serological diagnosis protocols.
Use of semiquantitative PCR to assess onset and treatment of Pneumocystis carinii infection in rat model
Authors: O'Leary T J , Tsai M M , Wright C F , Cushion M T
Published: January 2020

The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.
Detection of alpha- and beta-hemolytic-like activity from Campylobacter jejuni
Authors: Misawa N , Hirayama K , Itoh K , Takahashi E
Published: January 2020

Alpha-hemolytic-like activity from Campylobacter jejuni was clearly apparent when the medium pH ranged from 6.0 to 6.5, but the hemolytic zones disappeared when the pH of the medium increased. Beta-hemolytic-like activity just beneath the bacterial growth appeared after prolonged incubation. The hemolytic activity was not influenced by the species of blood.
A chemically defined liquid medium that supports primary isolation of Rochalimaea (Bartonella) henselae from blood and tissue specimens
Authors: Wong M T , Thornton D C , Kennedy R C , Dolan M J
Published: January 2020

Rochalimaea (Bartonella) henselae is a fastidious, slowly growing, gram-negative bacillus that is an etiologic agent of bacillary angiomatosis, cat scratch disease, and related syndromes. Accumulation of direct microbiologic evidence of the relationship between the organism and the syndromes compatible with cat scratch disease has been hindered by the difficulties in the primary isolation of the organism from infected tissue specimens. A chemically defined liquid medium was developed to support the growth of Rochalimaea species to facilitate study of the organism. This medium was also used successfully to isolate R. henselae from clinical specimens from infected patients and a domestic cat. Recovery of R. henselae in this was more successful than when recovery was attempted on solid agar. This cell-free, extract-free, defined medium additionally supported the growth of Rochalimaea quintana and Afipia felis.
Development of nested PCR based on the ViaB sequence to detect Salmonella typhi
Authors: Hashimoto Y , Itho Y , Fujinaga Y , Khan A Q , Sultana F , Miyake M , Hirose K , Yamamoto H , Ezaki T
Published: January 2020

For a rapid diagnosis of typhoid fever, we developed a nested PCR based on the nucleotide sequence encoding the Vi antigen. All Salmonella typhi strains along with a Salmonella paratyphi C strain were PCR positive. This assay was able to detect S. typhi at the single-cell level.
Zygospores: the last word in identification of rare or atypical zygomycetes isolated from clinical specimens
Authors: Weitzman I , Whittier S , McKitrick J C , Della-Latta P
Published: January 2020

Three zygomycetes (order Mucorales), isolated from clinical specimens, whose generic or species identification were uncertain, were definitively identified on the basis of the production of zygospores resulting from mating studies. These three isolates were identified as Mucor circinelloides f. circinelloides, Rhizomucor pusillus, and Absidia corymbifera. The production of true zygospores, the "last word" in zygomycete taxonomy, should be considered as a diagnostic tool for the definitive identification of rare, unusual, or atypical heterothallic zygomycetes. Practical considerations, however, limit this method to reference laboratories.
Burkholderia cepacia and cystic fibrosis: do natural environments present a potential hazard?
Authors: Butler S L , Doherty C J , Hughes J E , Nelson J W , Govan J R
Published: January 2020

An environmental survey of 55 sites yielded only 12 Burkholderia cepacia isolates, none of which displayed the phenotypic properties of a multiresistant epidemic strain associated with pulmonary colonization in patients with cystic fibrosis. Although the environment probably poses a low risk for patients with cystic fibrosis as a source of B. cepacia, the pathogenic potential of individual environmental strains remains unclear. We advise caution in the development of B. cepacia as a biocontrol agent.
Evaluation of lacto-phenol cotton blue for wet mount preparation of feces
Authors: Parija S C , Prabhakar P K
Published: January 2020

The use of lacto-phenol cotton blue (LPCB) stain for wet mount preparation of stools to demonstrate intestinal parasites by routine microscopy was evaluated in this study. LPCB-stained trophozoites and cysts and helminthic ova could easily be detected and identified in LPCB wet mounts of stools. The stain is recommended for routine use in the wet mount preparation of stools in a parasitology laboratory.
Detection of Borna disease virus RNA in formalin-fixed, paraffin-embedded brain tissues by nested PCR
Authors: Sorg I , Metzler A
Published: January 2020

A method for detecting Borna disease virus (BDV) RNA in formalin-fixed, paraffin-embedded brain tissue sections was established. By digestion with proteinase K and subsequent extraction with guanidinium thiocyanate, phenol, and chloroform, we were able to efficiently release RNA from the fixed tissues. By reverse transcription of the RNA and nested PCR a 212-bp product was generated, as expected.
Collagenases and the serine proteinases elastase and cathepsin G in steroid-induced Pneumocystis carinii pneumonia
Authors: Sukura A , Konttinen Y T , Sepper R , Kaartinen L , Sorsa T , Lindberg L A
Published: January 2020

Pneumocystis carinii pneumonia (PCP) is characterized by the formation of leaky alveoli and a foamy alveolar exudate. To induce PCP, male Wistar rats were immunosuppressed by oral dexamethasone treatment for 12 weeks, during which time all rats developed PCP. Bronchoalveolar lavage fluid (BALF) was analyzed at that time and at 1, 2, and 4 weeks after the cessation of dexamethasone treatment, during which time the rats were recovering from PCP and immunosuppression (and was compared with the BALF obtained from healthy control rats), for type IV collagenase, elastase, cathepsin G, and collagenase activities. Scores for 72-kDa (matrix metalloproteinase type [MMP-2]) and 92-kDa (MMP-9) type IV collagenase-gelatinase activities correlated with those for BALF macrophages (r = 0.58; P < 0.001) and neutrophils (r = 0.66; P < 0.001), respectively, suggesting that they may, in part, be derived from these cells. However, MMP-2 was constitutively expressed and may play a role in normal tissue remodeling. MMP-9 activity was highest in the group with PCP (1.8 +/- 0.37; P > 0.05), with a gradual decline (1.0 +/- 0.48 by week 4; P > 0.05) toward normal (0.67 +/- 0.42) during recovery, which suggests a role for it in tissue-destructive inflammatory events. In rats with PCP the endogenously active collagenase was present at high levels compared with those in healthy controls (2.6 +/- 0.69 versus 0.17 +/- 0.17, respectively; P < 0.01), but they returned to normal by week 4 of recovery (0.42 +/- 0.30; P > 0.05). Collagenase activity showed a correlation with cyst number (r = 0.57; P < 0.001). The BALF of rats with PCP also contained the serine proteinases, which may act as pro-MMP activators. Ultramorphology disclosed increased pinocytotic activities, subepithelial bleb formation, and degeneration and denudation of the basal lamina. These findings suggest that the increased activities of collagenases in BALF caused by the host response against P. carinii might contribute to leaky alveoli.
Evaluation and comparison of different blood culture techniques for bacteriological isolation of Salmonella typhi and Brucella abortus
Authors: Gaviria-Ruiz M M , Cardona-Castro N M
Published: January 2020

An experimental study was carried out to evaluate and compare various noncommercial methods of blood culture for the isolation of Salmonella typhi and Brucella abortus from fresh human blood samples that had been artificially inoculated with 1 to 50 microorganisms per ml of blood. The methods compared included the Ruiz-Castañeda blood culture, broth blood culture, leukocyte lysis and direct plating on agar (WBL-P), leukocyte lysis and filtration, Ficoll-Hypaque centrifugation and filtration, Ficoll-Hypaque centrifugation, and Ficoll-Hypaque centrifugation and leukocyte lysis methods. Results with the WBL-P technique showed that S. typhi was isolated in 18 h, and its recovery rate was 36.6% (calculated from the number of CFU recovered per milliliter versus the number inoculated). B. abortus was isolated in 48 h by the same technique, and its recovery rate was 48.8%. The isolation times for the other blood culture techniques were between 36 and 44 h for S. typhi and 66 h for B. abortus. The techniques which relied on filtering systems for the recovery of S. typhi and B. abortus performed poorly. The WBL-P technique for the isolation of S. typhi and B. abortus is faster than the other methods tested.
Identification and ribotypes of Staphylococcus caprae isolates isolated as human pathogens and from goat milk
Authors: Vandenesch F , Eykyn S J , Bes M , Meugnier H , Fleurette J , Etienne J
Published: January 2020

We report five cases of human infection with Staphylococcus caprae. Two were community acquired (one case each of endocarditis and urinary tract infection); the other three were acquired in a hospital (two cases of bacteremia associated with intravenous access and one case of urinary tract infection). Analysis of human isolates and goat isolates from eight herds showed that they could be misidentified by some commercial identification systems but were clearly identified as S. caprae by ribotyping, according to their species-specific ribotype. Phylogenetic methods applied to the ribotypes did not reveal two distinct lineages corresponding to the goat and human origins of the isolates, although human ribotypes were clearly distinguishable by the presence of a core of four specific bands. The latter observation may reflect some degree of evolutionary change within the species between human and goat isolates.
Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes
Authors: Jacobs M V , de Roda Husman A M , van den Brule A J , Snijders P J , Meijer C J , Walboomers J M
Published: January 2020

In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low risk (HPV-6, -11, -34, -40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6(+)-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.
Comparison of commercially available enzyme immunoassay with traditional serological tests for detection of antibodies to Coccidioides immitis
Authors: Martins T B , Jaskowski T D , Mouritsen C L , Hill H R
Published: January 2020

A newly released commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for its ability to detect immunoglobulin M (IgM) and IgG antibodies against the tube precipitin and complement fixation (CF) antigens of Coccidioides immitis. The ELISA was compared with more traditional diagnostic assays, CF, latex agglutination (LA), and immunodiffusion (ID). When the IgM-specific portion of the ELISA was compared with LA, there was an agreement of 81.8%, a specificity of 75.0%, and a sensitivity of 84.6%. For the determination of the presence of IgG antibodies, the results of the IgG-specific part of the ELISA were compared with the combined results of ID and CF. After resolution of discrepant results, there was an agreement of 95.6%, a specificity of 98.3%, and a sensitivity of 92.6%. When the results of the IgG- and IgM-specific portions of the ELISA combined were compared with the results of the three traditional assays (CF, LA, and ID) there was an agreement of 96.7%, a specificity of 98.5%, and a sensitivity of 94.8%. The ELISA proved to be a reliable assay for the detection of antibodies against the tube precipitin and CF antigens and did not suffer from the objectivity required to interpret the results of the traditional assays and anticomplement interference associated with the traditional assays.
Two markers, IS901-IS902 and p40, identified by PCR and by using monoclonal antibodies in Mycobacterium avium strains
Authors: Ahrens P , Giese S B , Klausen J , Inglis N F
Published: January 2020

The occurrence of two markers, a newly identified 40-kDa protein (p40) and the insertion sequence IS901-IS902, in strains of Mycobacterium avium subspp. was evaluated. Analysis of 184 type and field strains of the M. avium complex from human, animal, and environmental sources by PCR specific to IS901 and by a monoclonal antibody specific to p40 demonstrated the presence of the two molecular markers in all of the M. avium subsp. silvaticum strains examined and also in a number of M. avium subsp. avium strains (the latter isolated mainly from pigs). The appearance of the two markers was completely concurrent in all strains. Further, the marker-positive M. avium subsp. avium strains were mainly serotype 2, whereas M. avium complex strains of serotypes 4, 6, 8, 9, and 10 were marker negative. The M. avium subsp. avium type strains ATCC 25291 and approximately 50% of the M. avium subsp. avium field strains isolated from animals contained the markers, while only one strain of human origin was found to be marker positive. Therefore, IS901 and p40 appear to have substantial potential to differentiate among isolates of the M. avium complex. This observation raises new issues regarding classification of strains, since the presence of the markers was found to be inconsistent with the present taxonomic grouping of M. avium subspp.
Molecular epidemiology of Corynebacterium diphtheriae from northwestern Russia and surrounding countries studied by using ribotyping and pulsed-field gel electrophoresis
Authors: De Zoysa A , Efstratiou A , George R C , Jahkola M , Vuopio-Varkila J , Deshevoi S , Tseneva G , Rikushin Y
Published: January 2020

A selection of 100 Corynebacterium diphtheriae isolates from asymptomatic carriers and clinical cases from five regions in northwestern Russia were examined. Six additional isolates from patients in Finland and Estonia with epidemiological links to Russia were also examined. All isolates were characterized by biotyping, toxigenicity testing, ribotyping, and pulsed-field gel electrophoresis (PFGE). Hybridization of genomic DNA digested with BstEII revealed five ribotype patterns among the biotype gravis isolates (G1 through G5) and two patterns among the biotype mitis isolates (M1 and M2). PFGE using SfiI was not able to distinguish between ribotypes G1, G2, and G4. The predominant ribotype pattern, G1, found in cases of disease in all the areas studied, appears to be disseminating, in view of the isolates received from imported cases in Finland and Estonia. Among the 106 isolates examined, 68 produced pattern G1 and 24 produced pattern M1. Most of the M1 isolates were from the Leningrad Oblast region. Distinct ribotypes such as G2, G3, G4, G5, and M2 could represent endemic disease.
Comparison of automated Difco ESP blood culture system with biphasic BBL Septi-Chek system for detection of bloodstream infections in pediatric patients
Authors: Welby P L , Keller D S , Storch G A
Published: January 2020

We compared the Difco ESP 384 blood culture system with the pediatric Septi-Chek system for the detection of bloodstream infections in pediatric patients. A total of 10,762 blood culture sets included an ESP aerobic bottle and a Septi-Chek bottle. From these cultures, a total of 278 organisms classified as probable pathogens were isolated, including 237 from ESP bottles and 221 from Septi-Chek bottles. This difference was not statistically significant. More organisms classified as possible contaminants were also isolated from ESP bottles (for ESP, 480 bottles; for Septi-Chek, 418 bottles; P < 0.01). The time to detection was shorter for probable pathogens isolated from ESP bottles (median times for organisms isolated from both systems: ESP, 14.0 h; Septi-Chek, 34.5 h; P < 0.001). The proportions of all probable pathogens detected by 24 and 48 h after inoculation were 78 and 96%, respectively, for ESP compared with 31 and 74%, respectively, for Septi-Chek. The time to final identification was also shorter for organisms grown in ESP bottles (median times for organisms isolated from both systems: ESP, 48.8 h; Septi-Chek, 58.5 h; P < or = 0.001). A subset of 4,442 cultures also included an ESP anaerobic bottle in addition to an ESP aerobic bottle and a Septi-Chek bottle. There were no significant differences in the recovery of probable pathogens by any of the possible two bottle combinations, but five anaerobic pathogens were recovered only in the anaerobic bottle. We conclude that the ESP 384 is an excellent system for culturing pediatric blood samples and that it provides for the very rapid detection of bloodstream pathogens.
Differentiation of Campylobacter jejuni and Campylobacter coli strains by using restriction endonuclease DNA profiles and DNA fragment polymorphisms
Authors: Korolik V , Moorthy L , Coloe P J
Published: January 2020

The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolates from poultry and humans were analyzed by using DNA restriction endonucleases ClaI and EcoRV. The DNA restriction patterns produced by ClaI and EcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains of C. jejuni tested. The DNA restriction patterns were further examined by Southern blot analysis with a previously constructed DNA probe, pMO2005, which is also able to distinguish between C. jejuni and C. coli spp. (5). Two types of patterns were produced by hybridization with the ClaI-cleaved DNA of C. jejuni strains, one of a single 18.5-kb genomic fragment and the other of 14.5- and 4.0-kb fragments. This indicated the presence of an extra ClaI site in this genomic fragment in the strains with the duplex pattern. The Southern blot analysis of 169 C. jejuni and C. coli isolates from poultry and from humans with DNA probe pMO2005 demonstrated that 78% of C. jejuni strains isolated from chickens hybridized with DNA probe pMO2005 with a characteristic 14.5- and 4.0-kb banding pattern and 22% hybridized with a single 18.5-kb fragment, whereas 71% of human isolates hybridized with the single 18.5-kb fragment and only 29% hybridized with 14.5- and 4.0-kb fragments. These findings suggest that only a small proportion of C. jejuni strains that colonize chickens may cause disease in humans.
PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs
Authors: Mathis A , Deplazes P
Published: January 2020

A PCR assay for the diagnosis of leishmaniosis was developed by using primers that were selected from the sequence of the small-subunit rRNA gene. The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (lysis of erythrocytes in Tris-EDTA buffer, digestion with proteinase K directly in PCR buffer, and no further purification steps). Furthermore, an internal control is included in every specimen in order to detect the presence of PCR inhibitors. The PCR was compared with diagnostic in vitro cultivation of promastigote stages for the detection of Leishmania spp. in clinical specimens from humans and dogs with a tentative diagnosis of leishmaniosis. PCR and cultivation gave identical results with all but 1 of the 95 specimens from humans. The PCR result in this case was false negative, possibly because of unequal apportionment of this sample. With 10 skin biopsies from six patients with cutaneous leishmaniosis, the sensitivity was 60%. For six human immunodeficiency virus-positive patients with visceral leishmaniosis, all bone marrow biopsies and 7 of 11 whole blood samples (after isolation of leukocytes by Ficoll-Paque) were positive in both tests. PCR detected one more case with the use of 500 microliters of whole blood with direct lysis of the erythrocytes in Tris-EDTA buffer. With dog lymph node aspirates, the sensitivity was 100% (16 of 16 samples) for both methods; furthermore, PCR was positive for 5 of 13 whole blood samples from dogs with leishmaniosis. The specificity of the PCR was 100% (70 specimens from patients without leishmaniosis). This PCR assay proved to be feasible as a routine diagnostic test, being reliable and faster than in vitro cultivation.
Comparison of phenotypic characteristics, DNA-DNA hybridization results, and results with a commercial rapid biochemical and enzymatic reaction system for identification of viridans group streptococci
Authors: Kikuchi K , Enari T , Totsuka K , Shimizu K
Published: January 2020

The rapid ID 32 Strep system (bioMérieux, Marcy l'Etoile, France) was evaluated for its ability to identify 21 species of viridans group streptococci; results were compared with DNA-DNA hybridization results and results of conventional physiological tests. A total of 171 strains of the 21 species including 147 clinical strains was analyzed. Of the 156 strains of species included in the database of this system, 136 strains (87%) were correctly identified. Incorrect identification occurred for 13 strains (8%), and no identification was given for 7 strains (5%). It was difficult to differentiate S. mitis and S. oralis accurately with this system. Of the 17 strains identified as S. mitis by the rapid ID 32 Strep system, the results of DNA-DNA hybridization were in agreement for only 3 strains. S. crista and S. parasanguis, which are not included in the database, were identified as S. mitis or S. sanguis or were not identified, but S. parasanguis could probably be identified by using the rapid ID 32 Strep system because the biochemical profile is well characterized for this species. The rapid ID 32 Strep system can be used to differentiate most species for which phenotypic characteristics have been described if the database is revised according to recently reported amended criteria for the identification of viridans group streptococci. However, identification of a few species such as S. mitis and S. oralis is problematic with this system.
Genomic fingerprinting of epidemic and endemic strains of Stenotrophomonas maltophilia (formerly Xanthomonas maltophilia) by arbitrarily primed PCR
Authors: VanCouwenberghe C J , Cohen S H , Tang Y J , Gumerlock P H , Silva J
Published: January 2020

Arbitrarily primed PCR (AP-PCR) was used to type 64 clinical isolates of Stenotrophomonas maltophilia from 60 patients and the hands of one nurse. Forty-seven different patterns were observed, most patients having isolates with unique genomic fingerprints. A single pattern, however, was obtained from six of eight patients involved in an intensive care nursery outbreak, confirming the suspected nosocomial transmission of this microorganism. This strain was also found in four other patients hospitalized at the same time but in different units. AP-PCR typing results had a good correlation with the 49 patterns obtained when the isolates were typed by contour-clamped homogeneous electric field gel electrophoresis. Although AP-PCR is slightly less discriminatory than contour-clamped homogeneous electric field gel electrophoresis, it offers several advantages and should be considered as a practical option for molecular typing during investigations of outbreaks.
Comparison of E-test with broth microdilution and disk diffusion for susceptibility testing of coryneform bacteria
Authors: Martínez-Martínez L , Ortega M C , Suárez A I
Published: January 2020

The susceptibilities of 135 coryneform bacteria isolated from clinical samples to ampicillin (AMP), cephalothin (CR), cefoxitin (FOX), cefotaxime (CTX), erythromycin (E), ciprofloxacin (CIP), tetracycline (TE), amikacin (AK), vancomycin (VA), and rifampin (R) were determined by disk diffusion, broth microdilution, and the E-test. The following species (number of isolates in parentheses) were included: Corynebacterium urealyticum (30), Corynebacterium minutissimum (20), coryneform CDC group ANF-1 (20), Corynebacterium striatum (20), Corynebacterium jeikeium (15), coryneform CDC group I2 (8), Listeria monocytogenes (7), Corynebacterium xerosis (5), and other coryneform bacteria (10). Agreement within one twofold dilution between the E-test and broth microdilution was 31% (VA), 64% (AK), 71% (CTX), 77% (FOX and CIP), 79% (TE), 84% (AMP), 87% (E), and 88% (CR and R). For the 1,350 combinations of microorganisms and antimicrobial agents, 85 (6.3%) discrepancies in interpretive category were found (4.2% minor, 1.2% major, and 0.9% very major). Seventy (5.1%) disagreements in interpretive category were found between disk diffusion and the E-test (3.8% minor, 0.4% major, and 0.9% very major), and 85 (6.3%) disagreements were found between microdilution (reference method) and disk diffusion (4.2% minor, 0.5% major, and 1.5% very major). MICs obtained with the E-test were highly reproducible. No category discrepancy was observed for VA, despite quantitative results. Considering interpretive categories, there is a good overall agreement between the three methods studied here, but further evaluation of current methodologies for susceptibility testing is required when considering coryneform bacteria and determination of quantitative activity of antimicrobial agents.
Evaluation of disk method for hippurate hydrolysis by Campylobacter species
Authors: Nicholson M A , Patton C M
Published: January 2020

A disk method for hippurate hydrolysis was compared with the ninhydrin tube method by using 140 genetically confirmed Campylobacter strains. Results were similar for 129 (92%) strains when the inoculum size for the disk method was standardized. Six strains (4.2%) showed variable results by each method. Our results conflict with those obtained in studies by others, who found the two methods to be dissimilar.
Evaluation of a new, semiquantitative screening culture device for urine specimens
Authors: Ferguson J , Tanner J , Miller J M
Published: January 2020

The EZ Streak urine culture device (Difco Laboratories, Detroit, Mich.) combines the advantages of both the dip-slide and the classic urine culture technique, enabling bacterial enumeration and isolation following a simple inoculation step. Five hundred clean-catch urine specimens submitted by outpatients attending a health maintenance organization were used to compare the EZ Streak with culture using a 0.001-ml loop. Complete agreement of colony counts determined by the two methods was obtained for 114 (91.2%) of 125 positive specimens, but 99.2% of the results agreed in clinical interpretation, indicating the presence or absence of bacteriuria. Overall agreement between the EZ Streak and culture was 95.7%. The sensitivity and specificity of the EZ Streak were determined to be 98.4 and 99.4%, respectively. For this patient population, the EZ Streak was determined to be a reliable replacement for routine urine culture.
Use of restriction fragment length polymorphism as a genetic marker for typing Mycobacterium avium strains
Authors: Roiz M P , Palenque E , Guerrero C , Garcia M J
Published: January 2020

Restriction fragment length polymorphism (RFLP) was used to study 75 clinical isolates identified as Mycobacterium avium. Two repetitive insertion sequences, IS1311 and IS900, were used as DNA probes. Although less than 25% of isolates showed RFLP patterns with IS900, all strains gave banding patterns with IS1311. M. avium strains isolated from patients with AIDS exhibited marked polymorphism with both probes.
Identification of spirochetes isolated from wild rodents in Japan as Borrelia japonica
Authors: Masuzawa T , Suzuki H , Kawabata H , Ishiguro F , Takada N , Yano Y , Yanagihara Y
Published: January 2020

Spirochetes isolated from three species of wild mice, Apodemus speciosus, Apodemus argenteus, and Eothenomys smithi, which were caught on Honshu, Japan, were characterized on the bases of protein profiles, reactivities with monoclonal antibodies (MAbs), and DNA homology values. All the isolates reacted with MAb O1441b, which is specific for Borrelia japonica, but not with MAb P62, which is specific for Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. The DNA relatedness of four representatives isolates to strain HO14, a type strain of B. japonica, was 83 to 88%. From these findings, the spirochetes were identified as B. japonica.
Efficient discrimination of Mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified polymorphic DNA analysis
Authors: Abed Y , Davin-Regli A , Bollet C , De Micco P
Published: January 2020

Amplification of the region separating the genes coding for 16S and 23S rRNA was performed with 15 Mycobacterium tuberculosis isolates and the type strain, ATCC 27294. Reproducible amplification patterns were obtained. PCR products were then used as target DNA for random amplified polymorphic DNA (RAPD) analysis. The discriminatory power was higher than when whole genomic DNA was used as a RAPD template. 16S-23S spacer region-based RAPD analysis was a simple and efficient method of differentiation. Consequently, it may be a useful tool for epidemiologic studies of tuberculosis.
Evaluation of novel vancomycin-containing medium for primary isolation of Kingella kingae from upper respiratory tract specimens
Authors: Yagupsky P , Merires M , Bahar J , Dagan R
Published: January 2020

A new selective medium (BAV), consisting of trypticase agar with 5% sheep hemoglobin and 2 micrograms of vancomycin per ml, was compared with the routine blood-agar medium for the primary isolation of Kingella kingae from upper respiratory specimens from a population of young children. Infection was detected by the BAV medium in 43 of 44 (98%) cultures positive for K. kingae, and detection of the organism was facilitated by inhibition of gram-positive flora. Infection was detected in only 10 of 44 (23%) positive cultures by the blood-agar medium, and plates were usually covered by abundant normal flora, making the recognition of K. kingae much more difficult. Challenge of the medium with different organisms of respiratory origin showed that the BAV medium was inhibitory for gram-positive cocci and Haemophilus influenzae but that it supported growth of eight K. kingae strains isolated from patients with invasive infections. The new medium appears to be a useful epidemiological tool for studying the respiratory carriage of K. kingae.
Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing
Authors: Lockhart S R , Fritch J J , Meier A S , Schröppel K , Srikantha T , Galask R , Soll D R
Published: January 2020

The genetic homogeneity of nine commensal and infecting populations of Candida albicans has been assessed by fingerprinting multiple isolates from each population by Southern blot hybridization first with the Ca3 probe and then with the 0.98-kb C1 fragment of the Ca3 probe. The isolates from each population were highly related, demonstrating the clonal origin of each population, but each population contained minor variants, demonstrating microevolution. Variation in each case was limited to bands of the Ca3 fingerprint pattern which hybridized with the 0.98-kb C1 fragment. The C1 fragment was therefore sequenced and demonstrated to contain an RPS repetitive element. The C1 fragment also contained part or all of a true end of the RPS element. These results, therefore, demonstrate that most colonizing C. albicans populations in nonimmuno-suppressed patients are clonal, that microevolution can be detected in every colonizing population by C1 hybridization, and that C1 contains the repeat RPS element.
New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease
Authors: Wallace R J , Brown B A , Blacklock Z , Ulrich R , Jost K , Brown J M , McNeil M M , Onyi G , Steingrube V A , Gibson J
Published: January 2020

Nocardia brasiliensis, the second most frequently isolated aerobic actinomycete in the clinical laboratory, is usually associated with localized cutaneous infections. However, 22% of 238 N. brasiliensis isolates from the United States and 12% of 66 isolates from Queensland, Australia, which had been collected over a 17-year period, were associated with extracutaneous and/or disseminated diseases. Of the 62 invasive isolates, 37 (60%) were susceptible to ciprofloxacin and/or were susceptible to clarithromycin and resistant to minocycline, compared with only 6 (3%) of 242 localized cutaneous isolates. The 43 isolates with this susceptibility pattern appeared to define a new taxon. They were similar to Nocardia asteroides complex isolates clinically in proportions from persons with pulmonary (70%), central nervous system (23%), and/or disseminated diseases (37%) in the setting of corticosteroids (74%) or AIDS (14%). This putative new taxon differed from N. brasiliensis in the hydrolysis of adenine (92 versus 4%), beta-lactamase patterns on isoelectric focusing, and the presence of two early mycolic acid-ester peaks by high-performance liquid chromatography. Restriction analysis of a 439-bp fragment of the 65-kDa heat shock protein gene revealed that N. brasiliensis and the new taxon had different restriction patterns with 8 of the 11 enzymes tested. Screening of invasive isolates of N. brasiliensis for susceptibility to ciprofloxacin will identify most isolates of the new taxon, which likely represents a new Nocardia species.