bluuebunny/crossref_metadata_2025 huggingface dataset:
Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin
Authors: Shryock T R , White D W , Werner C S , Staples J M
Published: January 2020

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed.
Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice
Authors: Fox J G , Yan L L , Dewhirst F E , Paster B J , Shames B , Murphy J C , Hayward A , Belcher J C , Mendes E N
Published: January 2020

A fusiform bacterium with 3 to 14 multiple bipolar sheathed flagella and periplasmic fibers wrapped around the cell was isolated from the liver, bile, and lower intestine of aged, inbred mice. The bacteria grew at 37 and 42 degrees C under microaerophilic conditions, rapidly hydrolyzed urea, were catalase and oxidase positive, reduced nitrate to nitrite, did not hydrolyze indoxyl acetate or hippurate, and were resistant to both cephalothin and nalidixic acid but sensitive to metronidazole. On the basis of 16S rRNA gene sequence analysis, the organism was classified as a novel helicobacter, Helicobacter bilis. This new helicobacter, like Helicobacter hepaticus, colonizes the bile, liver, and intestine of mice. Although the organism is associated with multifocal chronic hepatitis, further studies are required to ascertain whether H. bilis is responsible for causing chronic hepatitis and/or hepatocellular tumors in mice.
Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal susceptibility testing
Authors: Colombo A L , Barchiesi F , McGough D A , Rinaldi M G
Published: January 2020

The use of Etest strips for antimicrobial susceptibility testing is a new and promising method with broad applications in microbiology. Since these strips contain a predefined continuous gradient of a drug, it is possible to obtain a reproducible, quantitative MIC reading. We performed a prospective and double-blinded study to compare the Etest and National Committee for Clinical Laboratory Standards (Villanova, Pa.) broth macrodilution methods for determining the MICs of fluconazole, itraconazole, and ketoconazole for 100 clinical isolates (25 Candida albicans, 25 Cryptococcus neoformans var. neoformans, 20 Torulopsis [Candida] glabrata, 15 Candida tropicalis, and 15 Candida parapsilosis). The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-P guidelines. Despite differences between results for some species-drug combinations, Etest and macrobroth MICs were, in general, in good agreement. The MIC agreement rates for the two methods, within +/- 1 dilution, were 71% for ketoconazole, 80% for fluconazole, and 84% for itraconazole. According to our data, Etest has potential utility as an alternative method.
Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR
Authors: Whelen A C , Felmlee T A , Hunt J M , Williams D L , Roberts G D , Stockman L , Persing D H
Published: January 2020

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.
Identification of vaccine-related polioviruses by hybridization with specific RNA probes
Authors: De L , Nottay B , Yang C F , Holloway B P , Pallansch M , Kew O
Published: January 2020

We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5'-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5'-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamant ane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.
Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis
Authors: Liveris D , Gazumyan A , Schwartz I
Published: January 2020

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.
Outbreak of Shigella sonnei infection traced to imported iceberg lettuce
Authors: Kapperud G , Rørvik L M , Hasseltvedt V , Høiby E A , Iversen B G , Staveland K , Johnsen G , Leitao J , Herikstad H , Andersson Y
Published: January 2020

In the period from May through June 1994, an increase in the number of domestic cases of Shigella sonnei infection was detected in several European countries, including Norway, Sweden, and the United Kingdom. In all three countries epidemiological evidence incriminated imported iceberg lettuce of Spanish origin as the vehicle of transmission. The outbreaks shared a number of common features: a predominance of adults among the case patients, the presence of double infections with other enteropathogens, and the finding of two dominant phage types among the bacterial isolates. In Norway 110 culture-confirmed cases of infection were recorded; more than two-thirds (73%) were adults aged 30 to 60 years. A nationwide case-control study comprising 47 case patients and 155 matched control individuals showed that the consumption of imported iceberg lettuce was independently associated with an increased risk of shigellosis. Epidemiological investigation of a local outbreak incriminated iceberg lettuce from Spain, consumed from a salad bar, as the source. The presence of shigellae in the suspected food source could not be documented retrospectively. However, high numbers of fecal coliforms were detected in iceberg lettuce from patients' homes. Three lettuce specimens yielded salmonellae. The imported iceberg lettuce harbored Escherichia coli strains showing resistance to several antimicrobial agents, including ampicillin, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole. During the outbreak it is likely that thousands of Norwegians and an unknown number of consumers in other countries were exposed to coliforms containing antibiotic resistance genes.
Specific detection of Brucella DNA by PCR
Authors: Romero C , Gamazo C , Pardo M , López-Goñi I
Published: January 2020

A PCR assay with primers derived from the 16S rRNA sequence of Brucella abortus was developed. Nine different combinations between six primers were tested. One pair of primers, which amplified a 905-bp fragment, was selected. As little as 80 fg of Brucella DNA was detected by this method. DNAs from all of the representative strains of the species and biovars of Brucella and from 23 different Brucella isolates were analyzed and yielded exclusively the 905-bp fragment. No amplification was detected with DNAs from 10 strains phylogenetically related to Brucella spp., 5 gram-negative bacteria showing serological cross-reactions with Brucella spp., and 36 different clinical isolates of non-Brucella species. Only Ochrobactrum anthropi biotype D yielded a PCR product of 905 bp, suggesting a closer relationship between Brucella spp. and O. anthropi biotype D. The specificity and high sensitivity of the PCR assay may provide a valuable tool for the diagnosis of brucellosis.
Inactivation of polymerase inhibitors for Mycobacterium tuberculosis DNA amplification in sputum by using capture resin
Authors: Amicosante M , Richeldi L , Trenti G , Paone G , Campa M , Bisetti A , Saltini C
Published: January 2020

Endogenous PCR inhibitors hamper DNA amplification of Mycobacterium tuberculosis DNA in sputum samples. Use of an anion binding resin (GeneReleaser [GR]) improved PCR sensitivity from 77% (27 of 35 culture-positive samples) with phenol-chloroform extraction (P-C) alone to 91% (GR without P-C; P > 0.05) and 100% (P-C and GR combined; P < 0.05).
Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization
Authors: Halonen P , Rocha E , Hierholzer J , Holloway B , Hyypiä T , Hurskainen P , Pallansch M
Published: January 2020

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.
Identification of varicella-zoster virus strains by PCR analysis of three repeat elements and a PstI-site-less region
Authors: Takada M , Suzutani T , Yoshida I , Matoba M , Azuma M
Published: January 2020

We established a method of identifying varicella-zoster virus (VZV) strains, especially those of the Oka vaccine, in patients with clinical VZV infections. The DNAs of 30 clinically isolated strains and 4 laboratory strains including the Oka vaccine strain and its parent VZV strain, were analyzed by PCR with four sets of primers for the four variable regions, R2, R4, R5, and a region without a PstI site (PS). R4 was unstable in four laboratory VZV strains and was excluded from the study. The other regions were stable in several passages in cell culture. The number of copies in R2 and R5 were distributed from 2 to 13 and from 1 to 3, respectively, in the strains analyzed. The vaccine strain had seven copies in R2 and two copies in R5, and it was PS negative. Among 30 clinical isolates, 3, 23, and 11 strains had the same characteristics as the vaccine strain in R2, R5, and PS, respectively. Therefore, by this method, 97.2% of the isolates were distinguished from the Oka vaccine strain. This strategy will be useful in diagnosing VZV infections induced by the vaccine strain.
Prosthetic valve endocarditis caused by Corynebacterium afermentans subsp. lipophilum (CDC coryneform group ANF-1)
Authors: Sewell D L , Coyle M B , Funke G
Published: January 2020

Corynebacteria are important causes of endocarditis in individuals with valvular prostheses. We report the first published case of prosthetic valve endocarditis caused by the newly defined species Corynebacterium afermentans subsp. lipophilum (former CDC coryneform group ANF-1). The isolate was recovered from a perivalvular abscess specimen and 5 of 15 Bactec blood cultures after 7 to 15 days of incubation. The isolation, identification, and susceptibility testing of Corynebacterium species are discussed.
Absence of human papillomavirus sequences in ovarian pathologies
Authors: Trottier A M , Provencher D , Mes-Masson A M , Vauclair R , Coutlée F
Published: January 2020

The role of human papillomaviruses (HPVs) in ovarian epithelium (either normal or neoplastic) remains controversial. We have investigated by PCR the presence of HPV DNA in fresh tissue from ovarian neoplasms and in primary cell cultures established from either normal, benign or malignant ovarian surface epithelia. None of the fresh samples and primary ovarian cultures contained HPV DNA sequences.
Mycobacterium asiaticum as the probable causative agent in a case of olecranon bursitis
Authors: Dawson D J , Blacklock Z M , Ashdown L R , Böttger E C
Published: January 2020

Mycobacterium asiaticum was isolated from fluid aspirated from an olecranon bursa that had become inflamed following a superficial injury. Other possible causes of the inflammation were excluded. No specific antimycobacterial therapy was given. The infection responded to drainage, regular dressing, and immobilization. Our experience suggests that M. asiaticum is a potential cause of infection of the joints and surrounding tissues.
Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid
Authors: West B , Wilson S M , Changalucha J , Patel S , Mayaud P , Ballard R C , Mabey D
Published: January 2020

A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible.
Detection of Rickettsia japonica in Haemaphysalis longicornis ticks by restriction fragment length polymorphism of PCR product
Authors: Uchida T , Yan Y , Kitaoka S
Published: January 2020

PCR was applied to the detection of Rickettsia japonica, the causative agent of Oriental spotted fever (OSF), in ticks collected at two sites of the Muroto area on Shikoku Island, a major area in Japan where OSF is endemic. Primer pair Rr190.70p and Rr190.602n of the R. rickettsii 190-kDa antigen gene sequence of Regnery and others (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) primed the DNA extracted from Haemaphysalis longicornis ticks but not those extracted from Haemaphysalis formosensis, Haemaphysalis flava, Haemaphysalis hystricis, or Amblyomma testudinarium ticks. Digestion of the amplification product with the restriction endonucleases PstI and AluI produced the restriction fragment length polymorphism pattern specific to R. japonica. The HindIII and MspI digests gave restriction fragment length polymorphism patterns identical to those of the PCR product from R. japonica DNA. Hemolymph preparations of H. longicornis ticks were demonstrated to contain rod-shaped organisms that were detected by immunofluorescence with the monoclonal antibody specific to R. japonica species. The primer pair did not amplify the DNA of a laboratory colony of H. longicornis ticks originally collected at an area where OSF is not endemic. Our results provided evidence that H. longicornis ticks might be an arthropod reservoir for R. japonica and a vector of OSF.
Detection and identification of aquatic birnaviruses by PCR assay
Authors: Blake S L , Schill W B , McAllister P E , Lee M K , Singer J T , Nicholson B L
Published: January 2020

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.
Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene
Authors: Mattsson J G , Bergström K , Wallgren P , Johansson K E
Published: January 2020

We report the use of PCR to detect DNA from Mycoplasma hyopneumoniae, the etiological agent of enzootic porcine pneumonia. A primer set was designed for the amplification of a 649-bp fragment of the 16S rRNA gene from M hyopneumoniae. The PCR product was identified by ethidium bromide staining after gel electrophoresis and by Southern hybridization with an M. hyopneumoniae-specific oligonucleotide probe. No amplification was observed from any other porcine bacteria tested, including several closely related mycoplasmas. It was also possible to demonstrate the presence of M. hyopneumoniae in bronchial lavage samples and lung tissue samples from experimentally infected pigs. Furthermore, the PCR system was used for analysis of nasal samples obtained from pigs in a fattening herd. By this method, we were able to detect M. hyopneumoniae in nose swabs from naturally infected pigs. However, our results suggest that M. hyopneumoniae can be detected in the nasal cavities only during a limited time period.
Parasitic culture of buffy coat for diagnosis of visceral leishmaniasis in human immunodeficiency virus-infected patients
Authors: López-Vélez R , Laguna F , Alvar J , Pérez-Molina J A , Molina R , Martinez P , Villarrubia J
Published: January 2020

Two samples of buffy coat from the peripheral blood of 25 human immunodeficiency virus-positive patients with proven visceral leishmaniasis, as determined with a bone marrow aspirate (stain and culture), were cultured onto Schneider's and Novy-McNeal-Nicolle media. Hemoculture positivity was 67%. The average growing time was 10 days. This is an easy, noninvasive, and sensitive technique.
Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood
Authors: Fujita S , Lasker B A , Lott T J , Reiss E , Morrison C J
Published: January 2020

We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.
Controlled study of Escherichia coli diarrheal infections in Bangladeshi children
Authors: Albert M J , Faruque S M , Faruque A S , Neogi P K , Ansaruzzaman M , Bhuiyan N A , Alam K , Akbar M S
Published: January 2020

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.
PCR for detection of rubella virus RNA in clinical samples
Authors: Bosma T J , Corbett K M , O'Shea S , Banatvala J E , Best J M
Published: January 2020

A reverse transcription nested PCR (RT-PCR) assay for the detection of rubella virus RNA using primers from the E1 open reading frame was established. This assay was found to be sensitive (detecting approximately two synthetic RNA copies and RNA extracted from 0.1 50% tissue culture infective dose of rubella virus) and specific; five wild-type rubella strains and four vaccine strains were detected, and no nonspecific amplification of 16 other RNA viruses or RNAs from seven cell types occurred. Rubella virus RNA was detected in 12 pharyngeal swabs from patients with serologically confirmed rubella; these RT-PCR results were in complete agreement with virus isolation. Analysis of products of conception obtained after confirmed primary maternal rubella infection by RT-PCR gave 92% agreement (12 of 13 samples) with virus isolation. No false-positive results were obtained. The potential use of this assay for prenatal diagnosis of congenital rubella infection and for investigating aspects of the pathogenesis of chronic disease is discussed.
Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia
Authors: Dumler J S , Asanovich K M , Bakken J S , Richter P , Kimsey R , Madigan J E
Published: January 2020

Homology in the 16S rDNAs shows that the agent of human granulocytic ehrlichiosis (HGE) is closely related to the veterinary pathogens Erlichia equi and Erlichia phagocytophila. After HGE, patients develop antibodies reactive with E. equi and E. phagocytophila; thus, we hypothesized that these species are closely related and share significant antigenicity. Antisera from humans, horses, dogs, and cattle were tested by indirect fluorescent-antibody assay (IFA) for antibodies reactive with E. equi and other ehrlichiae and tested by immunoblot to identify the specific reactions with E. equi. All convalescent-phase sera from human patients with HGE and from animals infected or immunized with E. equi or E. phagocytophila had antibodies reactive with E. equi by IFA; no reactions with Ehrlichia chaffeensis occurred with these sera, and only one horse naturally infected with E. equi had a serologic reaction against Ehrlichia sennetsu. Human and animal sera obtained after infection or immunization with other Ehrlichia, Rickettsia, and Bartonella species did not react with E. equi by IFA. E. equi immunoblots revealed as many as 19 bands with equine anti-E. equi serum. All HGE agent, E. equi, and E. phagocytophila antisera tested reacted with a 44-kDa antigen of E. equi, while other anti-Ehrlichia spp. sera reacted with this antigen rarely or not at all. HGE agent, E. equi, and E. phagocytophila antisera but not other sera also reacted occasionally with 25-, 42-, and 100-kDa antigens. Most sera reacted with antigens between approximately 56 and 75 kDa, probably heat shock proteins. The HGE agent, E. equi, and E. phagocytophila share significant antigenicity by IFA and immunoblot. Coupled with the nearly identical nucleotide sequences of 16S rRNA genes, these data indicate that E. equi, E. phagocytophila, and the human granulocytic ehrlichia are closely related or identical species.
Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine
Authors: Pfaller M A , Bale M , Buschelman B , Lancaster M , Espinel-Ingroff A , Rex J H , Rinaldi M G , Cooper C R , McGinnis M R
Published: January 2020

Amphotericin B, fluconazole, and flucytosine (5FC) were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Ten candidate QC strains were tested in accordance with National Committee for Clinical Laboratory Standards M27-P guidelines against the three antifungal agents in each of six laboratories. Each laboratory was assigned a unique lot of RPMI 1640 broth medium as well as a lot of RPMI 1640 common to all of the laboratories. The candidate QC strains were tested 20 times each against the three antifungal agents in both unique and common lots of RPMI 1640. A minimum of 220 MICs per drug per organism were generated during the study. Overall, 95% of the MICs of amphotericin B, fluconazole, and 5FC fell within the desired 3 log2-dilution range (mode +/- 1 log2 dilution). Excellent performance with all three drugs was observed for Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258. With these strains, on-scale 3 log2-dilution ranges encompassing 96 to 99% of the MICs of all three drugs were established. These two strains are recommended for QC testing of amphotericin B, fluconazole, and 5FC. Reference ranges were also established for an additional four strains for use in method development and for training. Four strains failed to perform adequately for recommendation as either QC or reference strains.
Evaluation of Staph ID 32 system and Staph-Zym system for identification of coagulase-negative staphylococci
Authors: Renneberg J , Rieneck K , Gutschik E
Published: January 2020

The purpose of the investigation was to evaluate two commercially available identification systems: a new modification of the Staph-Zym system (Rosco, Tåstrup, Denmark) and the Staph ID 32 API system (API System, BioMérieux, Paris, France). A local standard method to be used in routine laboratories was also evaluated. A total of 200 staphylococcal isolates, including strains from both the American Type Culture Collection and the Czechoslovak Collection of Microorganisms as well as 89 clinical isolates, were used in tests of all three identification systems. The Staph ID 32 API system identified from 50 to 100% of the reference strains and 82.1% of the clinical isolates correctly. The Staph-Zym system identified from 90 to 100% of the reference strains and 82.1% of the clinical isolates correctly. Most misidentifications were of minor importance, but in both systems major failures appeared (Staphylococcus aureus was identified as a coagulase-negative staphylococcus). Both systems needed backup from a reference laboratory to determine if two isolates were of the same strain.
Bovine vaginal antibody responses to immunoaffinity-purified surface antigen of Tritrichomonas foetus
Authors: Ikeda J S , BonDurant R H , Corbeil L B
Published: January 2020

Bovine trichomoniasis is a prevalent sexually transmitted disease of cattle caused by the protozoan Tritrichomonas foetus. Currently, diagnosis is most often made by culture. In order to provide a faster immunodiagnostic approach, a specific enzyme-linked immunosorbent assay (ELISA) was investigated. A protective surface antigen (TF1.17 antigen) of T. foetus was immunoaffinity purified and used in an ELISA to detect antibodies in vaginal mucus from heifers inoculated with T. foetus. In preliminary studies, antibodies of the immunoglobulin A (IgA) isotype were detected in mucus from all experimentally infected heifers which were tested at 6 weeks postinoculation, whereas IgG1 and IgG2 were not. In addition, IgA responses detected in postinoculation samples were all greater than those detected in preinoculation samples, unlike those detected by a whole-cell antigen ELISA. For these two reasons, IgA antibodies appeared to be useful diagnostically. Further investigation of IgA antibodies used vaginal mucus collected weekly from heifers inoculated intravaginally with 10(2), 10(4), or 10(6) T. foetus organisms. Heifers with positive cultures for T. foetus had similar IgA responses to TF1.17 antigen over the 10 weeks of infection regardless of the initial inoculum dose. This indicates that if the dose is sufficient to establish infection, the magnitude and duration of the immune response are no longer dependent on dose. All of the infected animals receiving all dosages responded with high absorbance values in the IgA anti-TF1.17 antigen ELISA by 6 weeks postinoculation, and all absorbance values remained high at 10 weeks. To determine the duration of the IgA response, four other heifers inoculated with 7 x 10(6) T. foetus organisms were studied through 24 weeks postinoculation. IgA antibody responses to TF1.17 antigen were still high at 24 weeks, even though some heifers cleared the infection several weeks earlier. These results indicate that experimentally infected heifers produced detectable long-lasting IgA responses to TF1.17 antigen in vaginal mucus, which may be useful in a herd test for the diagnosis of bovine trichomoniasis.
Molecular typing of Candida albicans in oral candidiasis: karyotype epidemiology with human immunodeficiency virus-seropositive patients in comparison with that with healthy carriers
Authors: Lupetti A , Guzzi G , Paladini A , Swart K , Campa M , Senesi S
Published: January 2020

Candida albicans organisms isolated from the oral cavities of healthy carriers (26 individuals) and compromised hosts (40 human immunodeficiency virus [HIV]-seropositive patients, all showing symptomatic oral candidiasis) were compared by resolving chromosome-sized DNA molecules into electrophoretic karyotypes. Seven- to 10-band electrophoretic patterns were obtained, with significant and reproducible differences in the distributions of the DNA bands. Seven distinct classes were identified and were designated type a (8 bands), type b (8 bands), type c (7 bands), type d (9 bands), type x (10 bands), type y (10 bands), and type z (9 bands). Four of these (types a to d) were the most representative within all of the isolated strains (95.5%), and the other three (types x to z) were observed only once in three HIV-seropositive individuals (4.5%). Only types b and c were isolated from healthy carriers, with the percentage of their isolation being 61.5 and 38.5%, respectively, while all the described karyotypes were isolated from HIV-seropositive patients, with type b being the most frequent (45%); this was followed by types c (25%), a (15%), and d (7.5%). The prevalence of type b and c karyotypes in HIV-infected individuals, as well as in healthy carriers, suggests that commensal strains in the oral cavities of healthy individuals may become the prevalent agents of subsequent oral candidiasis in compromised hosts. However, replacement of the original, commensal strain, if there is one, cannot be excluded in a compromised host, although strain replacement may be more reasonably hypothesized for types a and d, since only these types were isolated at a relative high percentage from the oral lesions of HIV-infected individuals.
Rapid identification of common human pathogens by high-resolution proton magnetic resonance spectroscopy
Authors: Delpassand E S , Chari M V , Stager C E , Morrisett J D , Ford J J , Romazi M
Published: January 2020

Routine procedures for recovery of bacteria from clinical specimens involve culturing the latter on various nonselective and selective agar media. The bacteria are then identified by means of biochemical and immunological test procedures. Reduction of the time required to identify the bacteria is highly desirable for rapid clinical diagnosis. Towards this end the potential of proton nuclear magnetic resonance (NMR) spectroscopy for providing a "fingerprint" within the proton spectrum of five bacterial genera, reflecting their characteristic cell wall constituents, has been investigated. Establishing a database of high-resolution proton NMR spectra of a large number of bacterial species is a prerequisite for attaining this objective. A database has been established for five common human pathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. On the basis of the presence of characteristic resonances in their spectra, a simple algorithm has been developed to differentiate and identify these microorganisms. The NMR spectra of E. coli and S. aureus showed no dependency on the type of growth medium, growth density, or incubation time.
Identification of Mycobacterium tuberculosis and M. avium complex directly from smear-positive sputum specimens and BACTEC 12B cultures by high-performance liquid chromatography with fluorescence detection and computer-driven pattern recognition models
Authors: Jost K C , Dunbar D F , Barth S S , Headley V L , Elliott L B
Published: January 2020

A high-performance liquid chromatography method that utilized fluorescence detection (HPLC-FL) of mycolic acid 6,7-dimethoxycoumarin esters was developed to identify Mycobacterium tuberculosis (MTB) and M. avium complex (MAC) directly from fluorochrome stain smear-positive sputum specimens and young BACTEC 12B cultures. HPLC-FL chromatograms from a training set that included 202 smear-positive clinical sputum specimens and 343 mycobacterial cultures were used to construct a calibrated peak-naming table and computer-based pattern recognition models for MTB and MAC. Pattern recognition model performance was measured with an evaluation set of samples that included 251 smear-positive clinical sputum specimens and 167 BACTEC 12B cultures. Evaluation sputum specimens were culture positive for MTB (n = 132) and MAC (n = 48). With evaluation sputa, the MTB and MAC models were 56.8 and 33.3% sensitive, respectively. Evaluation set BACTEC 12B cultures were culture positive for MTB (n = 97) and MAC (n = 53). The sensitivities of the MTB and MAC models for identification of BACTEC 12B cultures were 99.0 and 94.3%, respectively. The specificity of both models was 100% for both types of evaluation samples. The average times from BACTEC 12B inoculation to cell harvest were 10.2 and 7.4 days for MTB and MAC, respectively. HPLC-FL can identify MTB and MAC in 1 day from many smear-positive sputa. Rapid and sensitive identification of MTB and MAC from young BACTEC 12B cultures was achieved.
Antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy
Authors: McOrist S , Mackie R A , Lawson G H
Published: January 2020

Proliferative enteropathy is caused by the microaerophilic obligate intracellular bacterium ileal symbiont (IS) intracellularis. Treatment of this disease is problematic because of the lack of in vivo or in vitro data on the activities of antimicrobial agents. A new procedure for determining the susceptibility of IS intracellularis was developed by using a tissue culture system which promotes the in vitro multiplication of this organism. Nineteen antimicrobial agents were evaluated in triplicate cultures for their intracellular and extracellular activities against up to three IS intracellularis strains isolated from pigs with proliferative enteropathy. The MIC was defined as the lowest concentration which prevented multiplication of 99% of the IS intracellularis isolates. Penicillin, erythromycin, difloxacin, virginiamycin, and chlortetracycline were the most active compounds tested, all with MICs of < or = 1 microgram/ml. Tiamulin and tilmicosin were the next most active compounds, with MICs of < or = 4 micrograms/ml. The MICs of aminoglycosides were generally > 32 micrograms/ml. Both lincomycin and tylosin were relatively inactive against the IS intracellularis strains tested, with MICs of 32 and 64 micrograms/ml, respectively. These results indicate that some compounds capable of intracytoplasmic accumulation and blocking bacterial protein synthesis were active against IS intracellularis strains isolated from pigs with proliferative enteropathy. The in vitro cultivation system shows promise as a method for studying the interaction between IS intracellularis and antimicrobial agents and for screening new antibiotics for use in therapy.
Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene
Authors: Battles J K , Williamson J C , Pike K M , Gorelick P L , Ward J M , Gonda M A
Published: January 2020

Conserved primers were used to PCR amplify 95% of the Helicobacter hepaticus 16S rRNA gene. Its sequence was determined and aligned to those of related bacteria, enabling the selection of primers to highly diverged regions of the 16S rRNA gene and an oligonucleotide probe for the development of a PCR-liquid hybridization assay. This assay was shown to be both sensitive and specific for H. hepaticus 16S rRNA gene sequences.
Susceptibilities of 201 anaerobes to erythromycin, azithromycin, clarithromycin, and roxithromycin by oxyrase agar dilution and E test methodologies
Authors: Spangler S K , Jacobs M R , Appelbaum P C
Published: January 2020

The susceptibility of 201 anaerobes to erythromycin, azithromycin, clarithromycin, and roxithromycin was tested by agar dilution and E test methods by using a commercially available plate and dish system (OxyDish) to provide anaerobic conditions. Plates were incubated for 48 h. MICs for 50% of strains tested and MICs for 90% of strains tested by agar dilution and E test methods corresponded within 1 doubling dilution for all compounds. When all antibiotics were considered together, agar and E test MICs were within 1 and 2 doubling dilutions of each other in 84 to 91% and > 99% of cases, respectively.
Comparison of two rapid streptococcal antigen detection assays with culture for diagnosis of streptococcal pharyngitis
Authors: Heiter B J , Bourbeau P P
Published: January 2020

In this study, 801 pharyngeal specimens were cultured for group A streptococci and tested with the Biostar Strep A Optical Immunoassay (Strep A OIA). The respective sensitivities and specificities were as follows: culture, 97.1 and 100%; Strep A OIA, 91.5 and 94.8%. Of the 801 specimens, 597 were also tested with the Abbott TestPack Strep A Assay (TP-ST). For those specimens tested by all three methods, the respective sensitivities and specificities were as follows: culture, 98.1 and 100%; Strep A OIA, 92.3 and 95.4%; and TP-ST, 79.4 and 100%. The Strep A OIA is significantly more sensitive than TP-ST and compares favorably with culture.
Cell culture-PCR technique for detection of infectious cytomegalovirus in peripheral blood
Authors: Lipson S M , Ashraf A B , Lee S H , Kaplan M H , Shepp D H
Published: January 2020

We determined the efficacy of PCR for the direct detection of infectious cytomegalovirus (CMV) in specimen-inoculated MRC-5 tube cultures (cell culture-PCR [CC-PCR]). Parallel testing was performed by a shell vial assay-indirect immunofluorescence assay (SVA-IFA) and isolation (conventional MRC tube cultures [TC-CPE]. The sensitivity, specificity, and positive and negative predictive values for CC-PCR, SVA-IFA, and TC-CPE were 81, 99, 94, and 94%, 86, 100, 100, and 96%, and 77, 100, 100, and 93%, respectively. Future application of CC-PCR may be directed toward its use as a screening tool for cytomegalovirus genotypic variants.
Inter- and intraspecies identification of Bartonella (Rochalimaea) species
Authors: Roux V , Raoult D
Published: January 2020

Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp. Restriction fragment length polymorphism after PCR amplification of the 16S-23S rRNA gene ITS may be useful for rapid species identification, and PFGE could be an efficient method for isolate identification.