bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunomagnetic purification to facilitate DNA diagnosis of Plasmodium falciparum
Authors: Seesod N , Lunderberg J , Hedrum A , Aslund L , Holder A , Thaithong S , Uhlén M
Published: January 2020

The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the first time that immunomagnetic separation can be used to recover pathogens from whole blood and then used for PCR analysis. With antibodies to the merozoite surface protein (MSP1), the malaria-causing parasite Plasmodium falciparum was purified and concentrated from clinical samples. The recovered parasites were used directly for in vitro DNA amplification. The PCR product was subsequently analyzed by a colorimetric 96-well microtiter plate assay. The results from examining 117 patients attending a clinic in the Borai district, Thailand, demonstrate that the combined method with immunomagnetic separation followed by PCR increases the group of positively diagnosed patients compared with microscopic examination of stained blood films. Analysis of 1 microliter of whole blood resulted in a 12% (14 of 117) increase in positively diagnosed patients while a 10-microliters sample volume increased the positives diagnosed to 20.5% (24 of 117).
Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection
Authors: Grimprel E , Bégué P , Anjak I , Betsou F , Guiso N
Published: January 2020

Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study. The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B. pertussis in tests with other Bordetella species and other respiratory pathogens. The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults. The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions. In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively. In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively. PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies.
Utilization review of the use of BACTEC PLUS high-volume blood culture bottles
Authors: Porter R C , Lo P , Low D E , Simor A E , McGeer A , Scriver S , Moore T C , Goldman C , Skulnick M
Published: January 2020

The BACTEC PLUS 26 (NR26) (Becton Dickinson, Towson, Md.) high-volume blood culture bottle replaced the less expensive smaller-volume NR6A bottle in our hospital. An audit carried out several months after their introduction revealed that only 17.5% of the NR26 bottles received the required blood volume. Several audits and educational programs were required in order to achieve a compliance rate of > 60%.
Cytomegalovirus antigenemia: clinical correlations in transplant recipients and in persons with AIDS
Authors: Mazzulli T , Rubin R H , Ferraro M J , D'Aquila R T , Doveikis S A , Smith B R , The T H , Hirsch M S
Published: January 2020

We evaluated a rapid immunoperoxidase technique for the detection of cytomegalovirus (CMV) antigenemia in peripheral blood neutrophils of 56 transplant recipients (117 specimens) and 36 persons with AIDS (59 specimens). Antigenemia was 92% sensitive and 98% specific for the detection of clinical CMV infection in transplant recipients and 100% sensitive and 86% specific in persons with AIDS. Overall, CMV antigenemia was a more rapid and sensitive method for the detection of clinical CMV infection than either shell vial culture or conventional tube culture of blood.
Discriminatory power of typing schemes based on Simpson's index of diversity for Neisseria gonorrhoeae
Authors: Dillon J A , Rahman M , Yeung K H
Published: January 2020

Simpson's index of diversity was used to produce a single numerical value to compare the abilities of single or combined typing schemes to discriminate between unrelated isolates. This calculation was used to compare the discriminating power of auxotype and serovar determination and plasmid content analysis, either singly or in combination, for Neisseria gonorrhoeae isolates having different antimicrobial susceptibilities (i.e., antibiotic-susceptible isolates and those that produce penicillinase, carry plasmid-mediated resistance to tetracycline, have chromosomally mediated penicillin resistance, or both produce penicillinase and carry plasmid-mediated resistance to tetracycline). Plasmid content analysis and auxotype determination produced the lowest level of discrimination, while a combination of auxotype and serovar typing schemes generally provided higher levels of discrimination. Addition of plasmid content analysis to auxotype and serovar typing provided additional discrimination only with penicillinase-producing isolates. For isolates that carried plasmid-mediated resistance to tetracycline, isolates that were tetracycline resistant, isolates that both produced penicillinase and carried plasmid-mediated resistance to tetracycline, or isolates that had chromosomally mediated penicillin resistance, none of the typing methods produced high discriminatory indices, indicating that these isolates are probably derived from relatively few clones.
Use of the polymerase chain reaction with a murine model of picornavirus-induced myocarditis
Authors: Leparc I , Fuchs F , Kopecka H , Aymard M
Published: January 2020

Enteroviruses are common pathogens responsible for a wide spectrum of systemic infections. Conventional diagnosis of these infections relies on the isolation of viruses in cell culture and their identification by seroneutralization with polyclonal or monoclonal antibodies. Among enteroviruses, coxsackieviruses B have been involved as causative agents for viral myocarditis. Most of the time, in the case of cardiac pathologies, viral isolation is negative. Molecular biology techniques appear to be an alternative to conventional diagnosis and could supply evidence for the direct implication of enteroviruses in these severe pathologies. In this paper, we describe a murine experimental model of infection with the presumed highly cardiopathogenic coxsackie-virus B type 3. A kinetics of infection was observed for a period of 31 days, and the classical virological markers (viral isolation from feces and heart biopsies, seroconversion) were monitored and compared by means of molecular techniques (molecular hybridization, polymerase chain reaction [PCR]). In this 31-day period, the detection of coxsackievirus B type 3 RNA in the heart was possible only by using two successive seminested PCRs. After 9 to 11 days of active viral replication, when all other virological markers were negative, positive PCR signals were obtained, which supports the hypothesis of a shift to persistent enteroviral infection.
Candida albicans genotyping in studies with patients with AIDS developing resistance to fluconazole
Authors: Bart-Delabesse E , Boiron P , Carlotti A , Dupont B
Published: January 2020

We characterized Candida albicans strains responsible for recurrent oropharyngeal candidosis (OPC) in four patients with AIDS who developed clinical and mycological resistance to fluconazole (FCZ). Karyotype and restriction fragment length polymorphism analyses were performed on the clonal populations to differentiate relapse from reinfection, and the results were assessed with those of serotype and FCZ MICs. Despite the polymorphism in chromosomal bands larger than 2.2 Mbp related to an intraclonal variation, karyotype analysis showed a single strain type attributable to each patient. On the other hand, EcoRI and HinfI restriction fragments revealed a polymorphism for one patient between the first sample and the subsequent ones, relevant to the acquisition of a new strain causing the following episodes of OPC. This result coincided with switching of the serotype and with the acquisition of a resistance to FCZ. For the other three patients, the similarity of the DNA electrophoretic patterns and the serotype of the samples suggested that recurrence can be due to the initial strain that generates FCZ resistance. Although useful for epidemiological studies, molecular typing methods seem to be inadequate to detect the acquisition of FCZ resistance.
Detection of Mycobacterium leprae nasal carriers in populations for which leprosy is endemic
Authors: Klatser P R , van Beers S , Madjid B , Day R , de Wit M Y
Published: January 2020

In order to better understand the role of Mycobacterium leprae nasal carriage in the maintenance of infection reservoirs and transmission of leprosy, we applied a polymerase chain reaction (PCR) that detected a 531-bp fragment of the pra gene of M. leprae on nasal swab specimens collected through a total population survey from individuals living in an area in which leprosy is endemic. Among the total tested population of 1,228 people, 7.8% were found to be PCR positive. PCR positivity was shown to be randomly distributed among the population for which leprosy is endemic. No association was observed between PCR positivity, age, or sex. The observed distribution of PCR positivity among households of different sizes confirmed the expected values, with the exception of two households, each with three people with PCR-positive nasal swab specimens. Although nasal carriage does not necessarily imply infection or excretion of bacilli, the finding of nasal carriage supports the theory of a disseminated occurrence of M. leprae in populations for which leprosy is endemic.
Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction
Authors: Winters M A , Tan L B , Katzenstein D A , Merigan T C
Published: January 2020

Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in the plasma of seropositive individuals was performed by using an external control assay with techniques to standardize and control each measurement. Rigorous study of the variability of the assay showed that the median intraassay reproducibility was log10 0.15 RNA copies per ml of plasma, while the median interassay reproducibility on replicate plasma samples was log10 0.25 copies perml. Specimen stability studies showed reproducible recovery of RNA from plasma stored at -70 degrees C for up to 12 months. In clinically stable patients who were either untreated or taking zidovudine, the average week-to-week variation in plasma RNA levels, measured in real time, was log10 0.30 RNA copies per ml. In contrast, patients either initiating or changing antiretroviral therapy showed a fall of log10 0.8 to log10 2.0 copies per ml in plasma RNA levels. Overall, 105 of 110 (96%) HIV-1-seropositive individuals with CD4 counts of 36 to 868 cells per mm3 had quantifiable HIV-1 RNA over a range of log10 2.70 to log10 6.23 RNA copies per ml, including 81% (13 of 16) of the individuals with greater than 500 CD4 cells per mm3. Accurate and reproducible quantitation of plasma viremia in real time by reverse transcriptase polymerase chain reaction, particularly in asymptomatic HIV-1-infected individuals with high CD4 counts, provides a basis for the use of this virologic measure to monitor the short- and long-term effects of early intervention therapeutic strategies on viral burden.
Typing of strains from a single-source outbreak of Pseudomonas pickettii
Authors: Dimech W J , Hellyar A G , Kotiw M , Marcon D , Ellis S , Carson M
Published: January 2020

Plasmid profiles, genome restriction fragment polymorphisms, carbohydrate oxidation-fermentation reactions, methylumbelliferyl substrate hydrolysis patterns, antimicrobial susceptibilities, and results obtained with the Biolog GN biochemical substrate kit were used to type 19 common-source, but mixed-biotype, outbreak strains and one epidemiologically distinct strain of Pseudomonas pickettii. Biotyping with conventional and methylumbelliferyl substrates failed to distinguish between strains. Plasmid profile testing was found to be inconsistent and not reproducible. The Biolog GN kit allowed greater strain differentiation than restriction fragment polymorphism did (12 biotypes versus 5 biotypes); antimicrobial susceptibility testing yielded 4 biotypes, and oxidation-fermentation tests gave 3 biotypes. Oxidation-fermentation results were consistent with restriction fragment polymorphs in all but 1 of the 20 strains tested. For ease of typing, comprehensive typeability, and reproducibility, oxidation-fermentation tests should be performed initially and followed if necessary by restriction fragment polymorph analysis for the elucidation of P. pickettii infection outbreaks.
Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction
Authors: Paton A W , Paton J C , Goldwater P N , Manning P A
Published: January 2020

A single pair of oligonucleotide primers was used for polymerase chain reaction amplification of a 212- or 215-bp region of Escherichia coli Shiga-like toxin (SLT) genes from crude fecal culture extracts. Genes were typed by hybridization of the polymerase chain reaction products to SLT-I- or SLT-II-specific oligonucleotide probes. The procedure was capable of detecting fewer than 10 SLT-producing E. coli organisms per ml of culture against a background of more than 10(9) other organisms per ml and provides a rapid and sensitive means of screening primary fecal cultures for the presence of such strains. When this procedure was used to test primary cultures from gut contents or feces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT-II sequences.
Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California
Authors: Zingg B C , Brown R N , Lane R S , LeFebvre R B
Published: January 2020

Twenty-nine Borrelia burgdorferi isolates, obtained from dusky-footed wood rats (Neotoma fuscipes) and California kangaroo rats (Dipodomys californicus) in California, were analyzed genetically. Chromosomal DNA was examined by restriction endonuclease analysis (REA) and gene probe restriction fragment length polymorphism. Pulsed-field gel electrophoresis was used to analyze the plasmid profiles of the isolates. REA, the method with the greatest discrimination, disclosed 24 distinct restriction patterns among the 29 isolates. These restriction patterns were sorted into four restriction fragment length polymorphism groups on the basis of their gene hybridization patterns. Results of the REA and plasmid profile analysis supported this grouping. The degree of genetic diversity among Californian isolates demonstrated by our findings is greater than that previously reported among other groups of North American isolates and is similar or greater than the diversity reported among European isolates.
Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG), IgM, and IgA with broad specificity for enteroviruses
Authors: Swanink C M , Veenstra L , Poort Y A , Kaan J A , Galama J M
Published: January 2020

An antibody-capture enzyme-linked immunosorbent assay (ELISA) with coxsackievirus B1 as the antigen was evaluated for detection of immunoglobulin G (IgG), IgM, and IgA antibodies and showed broad specificity for enteroviruses. In total, 116 serum or cerebrospinal fluid samples from 62 patients were tested by ELISA and the complement fixation test (CFT). Additionally, 15 serum samples that contained poliovirus-specific IgM antibody were tested. Serum samples from 200 healthy blood donors were used for standardization of the assays. The sensitivity of the ELISA varied with time of serum sampling, with a relatively low sensitivity when serum was collected within 3 days after the onset of symptoms (23%; 5 of 22) but good sensitivity when serum was collected later (83%; 20 of 24). The sensitivity was better than that of the CFT. The ELISAs were broadly reactive as concluded from typing of virus isolates that were simultaneously obtained. The assay did, furthermore, detect antibody against poliovirus type 3. Sera that contained rheumatoid factor, antinuclear antibody, or cardiolipin antibody (by the Venereal Disease Research Laboratory test) did not react in this ELISA. Nonspecific reactivity did occur, however, in cases of infectious mononucleosis and in Mycoplasma pneumoniae infection. The enterovirus-specific ELISA is found to be simple to perform, more sensitive than the CFT, and far less laborious than the neutralization test.
Simultaneous detection of and differentiation between herpes simplex and varicella-zoster viruses with two fluorescent probes in the same test system
Authors: Brumback B G , Farthing P G , Castellino S N
Published: January 2020

Specimens from skin lesions were examined simultaneously for herpes simplex virus (HSV) and varicella-zoster virus (VZV) by direct specimen testing and shell vial culture in single-test systems. For direct testing, cells in a single specimen well were stained with a combination direct-indirect immunofluorescence stain by using two fluorescent tags. A total of 203 fresh specimens were tested in parallel. Of these, 100 specimens contained too few cells for the direct VZV comparison and 91 contained too few cells for the HSV comparison. After these specimens were eliminated, the sensitivities and specificities, respectively, of the dual direct test were 86.1 and 97.3% for HSV compared with single culture and 92.2 and 100% for VZV compared with single direct testing. Shell vial monolayers in the combined cultures were stained for both viruses by the same method. A total of 305 fresh specimens were cultured in parallel by dual- and single-culture methods. The sensitivities and specificities, respectively, of the combined culture compared with separate cultures were 100 and 98.4% for HSV and 87.9 and 99.2% for VZV. The combined methods gave a performance comparable to those of single tests, required less specimen volume, and were less costly to perform.
Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis
Authors: van Soolingen D , de Haas P E , Hermans P W , Groenen P M , van Embden J D
Published: January 2020

Five different genetic elements have been found to be associated with genetic rearrangements in Mycobacterium tuberculosis complex strains. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic marker for strain differentiation of M. tuberculosis. In the present study we compared five genetic elements for their potentials to differentiate a given cluster of M. tuberculosis strains. Because of the presence of only a single copy of IS6110 or two IS6110 copies at the same chromosomal locus, a large number of strains could not be differentiated by IS6110 fingerprinting. Most strains, including the low-copy-number IS6110 strains, could be differentiated by fingerprinting with the 36-bp direct repeat or the polymorphic GC-rich repetitive DNA element. Less discriminative power was obtained with the major polymorphic tandem repeat and the insertion element IS1081. One strain which did not contain IS6110 DNA was encountered. Until now, this element has invariantly been found to be present in all M. tuberculosis complex strains. On the basis of classical taxonomic criteria and sequencing of the 16S rRNA gene, this strain was shown to be a genuine M. tuberculosis strain. Therefore, the use of this element as a target for polymerase chain reaction-facilitated detection of M. tuberculosis should be reconsidered.
Simplified adsorption method for detection of antibodies to Candida albicans germ tubes
Authors: Ponton J , Quindos G , Arilla M C , Mackenzie D W
Published: January 2020

Two modifications that simplify and shorten a method for adsorption of the antibodies against the antigens expressed on both blastospore and germ tube cell wall surfaces (methods 2 and 3) were compared with the original method of adsorption (method 1) to detect anti-Candida albicans germ tube antibodies in 154 serum specimens. Adsorption of the sera by both modified methods resulted in titers very similar to those obtained by the original method. Only 5.2% of serum specimens tested by method 2 and 5.8% of serum specimens tested by method 3 presented greater than one dilution discrepancies in the titers with respect to the titer observed by method 1. When a test based on method 2 was evaluated with sera from patients with invasive candidiasis, the best discriminatory results (sensitivity, 84.6%; specificity, 87.9%; positive predictive value, 75.9%; negative predictive value, 92.7%; efficiency, 86.9%) were obtained when a titer of > or = 1:160 was considered positive.
Nosocomial epiglottitis associated with penicillin- and cephalosporin-resistant Streptococcus pneumoniae bacteremia
Authors: Daum R S , Nachman J P , Leitch C D , Tenover F C
Published: January 2020

We cared for a 4-year-old male with nosocomially acquired epiglottitis caused by Streptococcus pneumoniae. He had been receiving ceftazidime therapy when this infection was recognized. The S. pneumoniae isolate was of serotype 15B and was resistant to beta-lactam antibiotics, cephalosporins (including those with extended spectra), and trimethoprim-sulfamethoxazole. Clinicians and clinical microbiologists must be aware that cephalosporin susceptibility may no longer be assumed for penicillin-resistant S. pneumoniae isolates and that susceptibility testing for the extended-spectrum cephalosporins should be performed whenever this species is isolated from a normally sterile body fluid.
PCR-based random amplified polymorphic DNA fingerprinting of Yersinia pseudotuberculosis and its practical applications
Authors: Makino S , Okada Y , Maruyama T , Kaneko S , Sasakawa C
Published: January 2020

The random amplified polymorphic DNA (RAPD) fingerprinting method was used to distinguish between various strains of Yersinia pseudotuberculosis, a causative agent of gastroenteritis in humans. The RAPD test uses arbitrarily chosen DNA oligomers of 10 nucleotides to prime DNA synthesis from genomic sites to which they are accidentally matched or almost matched. Most 10-nucleotide primers yielded strain-specific arrays. Ten Y. pseudotuberculosis type strains were distinguishable from each other by analyzing the RAPD arrays produced by using primers with a 50% G+C content. The RAPD patterns of Y. pseudotuberculosis strains were found to be constant regardless of the presence or absence of the large plasmid. RAPD tests were subsequently used to identify 30 clinical isolates of Y. pseudotuberculosis that were collected as the causative agent of an outbreak of Izumi fever, a disease showing clinical symptoms characteristic of atypical scarlet fever, in Japan. The RAPD arrays from all of the isolates yielded common patterns that were unique to each primer used. Since those 30 isolates belonged to serotype 5a and the restriction digest patterns of their large plasmids were all the same, the results of the RAPD tests confirmed the view that those isolates were from a single source and indicated that the RAPD test can be practically applied to survey transmission of the bacterium in humans.
Identification and characterization of genetic cluster groups of Actinobacillus actinomycetemcomitans isolated from the human oral cavity
Authors: DiRienzo J M , McKay T L
Published: January 2020

Actinobacillus actinomycetemcomitans is recognized as a primary pathogen in localized juvenile periodontitis (LJP). Restriction fragment length polymorphisms (RFLP) within a collection of subgingival plaque isolates of this bacterium were identified and characterized as the first step in understanding the pathogenesis of LJP. Over 800 isolates, from members of 18 families (LJP families) with at least one member with active LJP or a documented history of the disease and one or more siblings, less than 13 years of age, having no clinical evidence of LJP and 32 healthy control subjects, were assigned to one of 13 distinct RFLP groups (II to XIV) by using a previously characterized 4.7-kb DNA probe cloned from the reference strain FDC Y4. Isolates belonging to RFLP groups II, IV, V, and XIII predominated subgingival sites in the subjects. Members of RFLP groups II, IV, VII, VIII, X, and XI were recovered only from LJP family subjects, while group XIII and XIV variants were found exclusively in healthy controls. A synthetic oligonucleotide, homologous to the 5' end of the leukotoxin gene (lktA), and the A. actinomycetemcomitans plasmid, pVT745, were tested for their abilities to subdivide the 13 RFLP groups. The leukotoxin probe specifically identified all RFLP group II variants because of the absence of a HindIII site in the upstream noncoding region of the lkt gene complex. The plasmid probe was not as selective but may be useful for identifying clinical isolates belonging to RFLP group I. The use of these probes for the identification of genetic variants of A. actinomycetemcomitans that may be preferentially colonize diseased and healthy subjects will facilitate the study of the role of this important pathogen in periodontal diseases.
Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis
Authors: Skuce R A , Brittain D , Hughes M S , Beck L A , Neill S D
Published: January 2020

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries.
Utility of complement fixation and microimmunofluorescence assays for detecting serologic responses in patients with clinically diagnosed psittacosis
Authors: Wong K H , Skelton S K , Daugharty H
Published: January 2020

The serodiagnosis of human psittacosis was considerably improved by a microimmunofluorescence (MIF) assay that uses selected strains of Chlamydia psittaci, C. pneumoniae, and C. trachomatis as antigens. The 78 patients examined in the study were clinically diagnosed as having psittacosis on the basis of compatible clinical symptoms following exposure to sick birds. The conventional complement fixation (CF) test identified 36 patients, or 46% (36 of 78) of the total, as positive. Antibody responses to C. psittaci were demonstrated by the MIF test in all 36 CF-positive patients. The MIF test also detected antibody responses to C. psittaci in 12 patients (15% of the total) whose sera were negative or anticomplementary in the CF test. Seven patients, or 9% (7 of 78) of the total, were identified by the MIF test as having C. pneumoniae infections. About 30% of the study patients (23 of 78) showed no serologic evidence of either C. psittaci or C. pneumoniae infection by both the CF and the MIF tests. Four distinctive serologic reaction patterns were observed in the study patients. Recognition of these reaction patterns and judicious corroboration of serologic responses to the chlamydial species by the MIF test with epidemiologic and clinical information will increase the efficiency and accuracy of serodiagnosis for human psittacosis.
Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis
Authors: Tebourski F , el Gaied A , Louzir H , Ben Ismail R , Kammoun R , Dellagi K
Published: January 2020

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.
Preliminary study using species-specific oligonucleotide probe for rRNA of Bilophila wadsworthia
Authors: Sapico F L , Reeves D , Wexler H M , Duncan J , Wilson K H , Finegold S M
Published: January 2020

Portions of the 16S RNA from a urease-positive Bilophila wadsworthia strain were sequenced, and a probe was constructed. The probe was end labeled with [32P]ATP and polynucleotide kinase and hybridized on a nylon filter (by dot blot hybridization) to the immobilized rRNA of 12 B. wadsworthia strains and eight other anaerobic isolates. The probe efficiently hybridized only to the Bilophila strains. Cross-reactivity at high RNA levels (2,000 ng) was observed with one strain of Bacteroides thetaiotamicron and one strain of Bacteroides fragilis (with 10x SET buffer [20x SET buffer is 0.5 M NaCl, 0.03 M Tris, and 2 mM EDTA]) but was not seen at lower RNA levels or with 5x SET buffer. When tested against mixed cultures of aerobic and anaerobic isolates representative of appendiceal abscess flora, the probe did not react with mixed cultures containing no Bilophila cells and could detect > or = 10(5) Bilophila CFU/ml when the mixture was seeded with Bilophila cells. This probe is of potential use in the rapid identification of pure isolates and in the direct identification of B. wadsworthia in clinical specimens.
Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals
Authors: Arlet G , Rouveau M , Casin I , Bouvet P J , Lagrange P H , Philippon A
Published: January 2020

Preliminary results suggested that the diffusion in France of the SHV-4 extended-spectrum beta-lactamase was probably due to the spread of one single epidemic strain of Klebsiella pneumoniae. In this study, we tested various phenotypic and genotypic markers to compare K. pneumoniae strains producing this enzyme isolated in 14 French hospitals between 1987 and 1989. All of the strains were of the same capsule serotype, K25. Twelve of them were of the same biotype: weak urease activity and no sucrose fermentation. Among the six plasmid profiles observed, one accounted for eight strains. Large plasmids of 170 kb encoding SHV-4 beta-lactamase were present in all strains of K. pneumoniae and could be transferred by conjugation with high frequency to Escherichia coli J53-2 or HB101 from all except one strain. Plasmid EcoRI restriction patterns suggested that these plasmids were closely related and similar to pUD18 encoding SHV-3 beta-lactamase, originally described in France and differing from SHV-4 by one amino acid substitution. Ribotyping with EcoRI and HindIII and genomic fingerprinting with XbaI by pulsed-field gel electrophoresis were concordant and suggested that 12 of the isolates recovered from the 14 hospitals were probably the same strain. Dissemination in France of the SHV-4 extended-spectrum beta-lactamase was thus essentially due to the diffusion of a single K. pneumoniae clone.
Evaluation of a new reagent for anti-cytomegalovirus and anti-Epstein-Barr virus immunoglobulin G
Authors: Gutierrez J , Maroto M D , Piédrola G
Published: January 2020

The Enzygnost alpha method was tested against the complement fixation test and anti-VCA immunofluorescence to determine the respective titers of anti-cytomegalovirus and anti-Epstein-Barr virus immunoglobulin G antibodies. For cytomegalovirus, the Enzygnost results showed 97.99% agreement with the readings obtained by the alternative method, with 100% sensitivity and 93.7% specificity. For Epstein-Barr virus, Enzygnost showed 97.71% agreement, 100% sensitivity, and 91.11% specificity.
Diagnosis of cytomegalovirus infections by shell vial assay and conventional cell culture during antiviral prophylaxis
Authors: Mañez R , St George K , Linden P , Martin M , Kusne S , Grossi P , Ho M , Rinaldo C
Published: January 2020

A total of 3,552 specimens for conventional cytomegalovirus (CMV) culture and shell vial assay for CMV immediate-early antigen were obtained during a prospective randomized trial for prophylaxis of CMV disease after liver transplantation. Prophylaxis with ganciclovir for 2 weeks and then high-dose acyclovir for 2.5 months was compared with high-dose acyclovir alone for 3 months. During the first 12 weeks after transplantation, when the patients were on prophylaxis, there were significantly more clinical samples positive by the shell vial assay and negative by standard culture in comparison with the number of samples obtained from weeks 13 to 24, after prophylaxis was discontinued, that were positive by the shell vial assay and negative by standard culture. In contrast, significantly fewer samples were positive by both the shell vial assay and standard culture during the first 12 weeks compared with the number obtained 13 to 24 weeks after transplantation that were positive by both methods. Samples positive by the shell vial assay only were obtained significantly more frequently from patients with asymptomatic than symptomatic CMV infections, while samples positive by both methods were obtained significantly more often from patients with symptomatic CMV infection. It was concluded that antiviral prophylaxis with high-dose acyclovir or ganciclovir and then high-dose acyclovir and asymptomatic CMV infection are associated with a decrease in the level of CMV isolation by standard cell culture in comparison with that by the shell vial assay.
Disk diffusion versus broth microdilution susceptibility testing of Haemophilus species and Moraxella catarrhalis using seven oral antimicrobial agents: application of updated susceptibility guidelines of the National Committee for Clinical Laboratory Standards
Authors: Kibsey P C , Rennie R P , Rushton J E
Published: January 2020

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 beta-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillin-clavulanic acid. beta-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.
Evaluation and characterization of multiresistant Enterococcus faecium from 12 U.S. medical centers
Authors: Sader H S , Pfaller M A , Tenover F C , Hollis R J , Jones R N
Published: January 2020

Forty-two Enterococcus faecium isolates resistant to ampicillin, penicillin, gentamicin, streptomycin, vancomycin, and teicoplanin (VanA phenotype) from 12 U.S. medical centers were analyzed by pulsed-field gel electrophoresis of chromosomal DNA. The isolates were tested for susceptibility to 12 alternative drugs. The results indicated both intrahospital and interhospital diversity among multiresistant vanA enterococcal isolates. Furthermore, the finding of isolates with identical pulsed-field gel electrophoresis patterns in different centers strongly suggests some interhospital clonal transmission.
Pneumonia caused by Stenotrophomonas maltophilia with a mucoid phenotype
Authors: Irifune K , Ishida T , Shimoguchi K , Ohtake J , Tanaka T , Morikawa N , Kaku M , Koga H , Kohno S , Hara K
Published: January 2020

We describe the first known case of pneumonia caused by a mucoid Stenotrophomonas maltophilia (Xanthomonas maltophilia) strain in a patient with bronchiectasis. The patient was admitted because of mild hemoptysis and productive cough with infiltrative shadow in the right lower lung field on chest X ray. The clinical symptoms were mild, and treatment with minocycline was effective.
Genetic variability among Chlamydia trachomatis reference and clinical strains analyzed by pulsed-field gel electrophoresis
Authors: Rodriguez P , Allardet-Servent A , de Barbeyrac B , Ramuz M , Bebear C
Published: January 2020

Pulsed-field gel electrophoresis (PFGE) was applied to Chlamydia trachomatis reference strains representing each of the 18 serovars and to 29 clinical isolates from genital specimens collected in Bordeaux, France, or Malmö, Sweden. Comparison of the fingerprint patterns of the reference strains revealed a high level of polymorphism of the total DNA when SmaI was used (14 profiles), whereas the other enzymes, Sse8387I and ApaI, showed fewer differences. Some serovars, considered to be closely related on the basis of their antigenic determinants located on the major outer membrane protein (MOMP), such as D and Da or I and Ia, were shown to be different after PFGE of their genomic DNAs. However, serovars B and Ba and serovars L2 and L2a had identical patterns after analysis with the three endonucleases. When applied to clinical isolates, which were typed by restriction fragment length polymorphism analysis of the MOMP gene, PFGE allowed the detection of intragenotype polymorphisms and showed the identity of two strains successively isolated from the same patient. This technique seems to be an efficient tool for epidemiological studies when used in addition to serotyping or genotyping by restriction fragment length polymorphism analysis of the MOMP gene.
Identification of mycobacteria by PCR-based sequence determination of the 32-kilodalton protein gene
Authors: Soini H , Böttger E C , Viljanen M K
Published: January 2020

In this study, a part of the nucleotide sequence of the mycobacterial 32-kDa protein gene was determined by PCR-based sequencing. A total of 24 mycobacterial strains, representing 10 species, were studied. Sequences of all tested members of the Mycobacterium tuberculosis complex were identical to each other and to the previously published sequence of M. tuberculosis H37Rv. The sequences of M. avium and M. intracellulare were different from each other. MAIX strains, identified with the Gen-Probe MAIX test, had sequences identical to each other but clearly different from those of M. avium and M. intracellulare. Each of the other mycobacterial species investigated, i.e., M. kansasii, M. gastri, M. gordonae, and M. malmoense, had a unique species-specific sequence. These results demonstrate that there is variation in the nucleotide sequence of the 32-kDa protein gene among different mycobacterial species. Thus, we propose that this gene can be used for PCR-based identification of mycobacteria.
Detection of Borrelia burgdorferi sensu lato in lesional skin of patients with erythema migrans and acrodermatitis chronica atrophicans by ospA-specific PCR
Authors: Moter S E , Hofmann H , Wallich R , Simon M M , Kramer M D
Published: January 2020

The aim of this study was to develop a sensitive and specific PCR for the detection of Borrelia burgdorferi DNA. The plasmid-located gene coding for the outer surface protein A (OspA [31-kDa protein]) was used as a target. Nucleotide sequence information from different B. burgdorferi ospA genotypes was used to design primers homologous to different genotypes. The sensitivity of the nested PCR differed from 1 fg to 1 pg of borrelial DNA, depending on the strain analyzed. No cross-reactions with DNA from spirochetes other than B. burgdorferi or with human DNA were observed. A total of 22 skin biopsy samples from patients with erythema migrans (EM [n = 10]) or acrodermatitis chronica atrophicans (ACA [n = 12]) were examined for the presence of B. burgdorferi by nested PCR. Of 22 biopsies, 80% from EM patients and 92% from ACA patients were positive by PCR amplification. By comparison, 50% of the EM patients had elevated B. burgdorferi-specific immunoglobulin M (IgM) and/or IgG antibody levels as tested by enzyme-linked immunosorbent assay (ELISA) using purified B. burgdorferi flagella as antigen. A total of 33% of ACA patients had elevated IgM titers, and all had high IgG titers in their sera. Only 30% of specimens from patients with EM and none from patients with ACA were positive by culture. All culture-positive specimens were also positive by PCR. Thus, the sensitivities of the PCR were 80 and 92%, respectively, for patients with EM and ACA on the basis of the clinical and histopathological diagnoses of Lyme disease. From these results, we conclude that PCR is a suitable method to detect B. burdorferi sensu lato DNA in skin biopsy samples and could be applied as an additional diagnostic tool.
Restriction fragment length polymorphism Mycobacterium tuberculosis strains isolated from Greenland during 1992: evidence of tuberculosis transmission between Greenland and Denmark
Authors: Yang Z H , de Haas P E , van Soolingen D , van Embden J D , Andersen A B
Published: January 2020

In order to describe the transmission of tuberculosis (TB) at the clonal level in a defined geographic region during a certain period of time, all isolates of Mycobacterium tuberculosis collected during 1992 from Greenland were subjected to analyses of DNA restriction fragment length polymorphism (RFLP). The RFLP patterns obtained by probing the genomic DNA with the repetitive insertion segment IS6110 revealed a high degree of similarity among the isolates, indicating a relatively high transmission rate and a close relationship between the individual M. tuberculosis clones. This was further confirmed by reprobing the Southern blots with two more-stable genetic markers, IS1081 and the DR sequence. The RFLP patterns were compared with those of 245 M. tuberculosis strains collected from Denmark during the same period (representing 91% of all new, bacteriologically verified cases of TB in Denmark in 1992). One of the three prevalent IS6110-defined clusters was traced to a group of immigrants from Greenland living in a small, defined geographical region in Denmark and to a group of Danish citizens either with known contact with these immigrants or, in other cases, with a record of previous travel or working activities in Greenland. The study showed that the present technique is extremely helpful in monitoring the spread of TB and thereby also contributing to improved disease control.
Helicobacter sp. strain Mainz isolated from an AIDS patient with septic arthritis: case report and nonradioactive analysis of 16S rRNA sequence
Authors: Husmann M , Gries C , Jehnichen P , Woelfel T , Gerken G , Ludwig W , Bhakdi S
Published: January 2020

A campylobacter-like organism was isolated from an effusion of the left knee joint of an AIDS patient 2 weeks after bacteremia with a morphologically identical organism. Amplified genomic 16S rRNA sequences were analyzed by a nonradioactive blotting technique. The closest match was found with Helicobacter fenelliae (97.7% homology). Sequence data and phenotype suggest that the isolate may represent a so far unrecognized species of the genus Helicobacter.
Subtype analysis of hepatitis C virus in Indonesia on the basis of NS5b region sequences
Authors: Hotta H , Handajani R , Lusida M I , Soemarto W , Doi H , Miyajima H , Homma M
Published: January 2020

A recently identified subtype of hepatitis C virus, subtype 1d, was found to be common in Indonesia, being isolated from 4 (20%) of 20 and 11 (34%) of 32 patients with chronic hepatitis and liver cirrhosis, respectively. A new group of sequence variants was also identified, although its prevalence ratio was not as high in the area surveyed.
Genomic Stability of
Legionella pneumophila
Isolates Recovered from Two Cardiac Transplant Patients with Nosocomial Legionnaires' Disease
Authors: Marrie Thomas J. , Johnson Wendy M. , Tyler Shaun D. , Bezanson Gregory S. , Burbridge Susan
Published: January 2020

Pulsed-field gel electrophoresis revealed that multiple consecutive isolates of
Legionella pneumophila
from two cardiac transplant patients remained genomically stable, despite exposure to host defenses and antimicrobial agents.
Case of Aeromonas veronii (DNA group 10) bacteremia
Authors: Abbott S L , Serve H , Janda J M
Published: January 2020

We describe the first case report of bacteremia due to Aeromonas veronii biotype veronii. The infection occurred in a 77-year-old man suffering from multiple underlying conditions which included cancer of the sigmoid colon. Because of the unusual biochemical phenotype of this group (ornithine decarboxylase positive), it was originally identified as Vibrio cholerae.