bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular study of persistence of Nocardia asteroides and Nocardia otitidiscaviarum strains in patients with long-term nocardiosis
Authors: Provost F , Laurent F , Uzcategui L R , Boiron P
Published: January 2020

Three Nocardia asteroides and five Nocardia otitidiscaviarum isolates recovered from three patients with long-term nocardiosis were compared by random amplification of polymorphic DNA fingerprinting, antibiotic susceptibility testing, and enzymatic characterization. Results obtained with primer DKU49 (P. Palittapongarnpim, S. Chomyc, A. Fanning, and D. Kunimoto, J. Infect. Dis. 167:975-978, 1993) provide evidence that patient A was infected by two N. asteroides strains during a single episode of nocardiosis and that patients B and C remained infected by the same strain, respectively. Resistance to minocycline that was present in the first isolate recovered from patient B reverted to intermediate resistance in the second isolate and reverted to susceptibility in the third isolate. Resistance to penicillin G and beta-lactams was acquired by the second isolate obtained from patient C.
Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis
Authors: Kolbert C P , Bruinsma E S , Abdulkarim A S , Hofmeister E K , Tompkins R B , Telford S R , Mitchell P D , Adams-Stich J , Persing D H
Published: January 2020

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.
Detection of Colorado tick fever virus by using reverse transcriptase PCR and application of the technique in laboratory diagnosis
Authors: Johnson A J , Karabatsos N , Lanciotti R S
Published: January 2020

Colorado tick fever (CTF) virus elicits an acute illness in humans, producing nonspecific flu-like symptoms and a biphasic fever in approximately 50% of patients. The disease is transmitted by the adult Rocky Mountain wood tick (Dermacentor andersoni), and therefore incidence is limited by the habitat and life cycle of that vector. The early symptoms of infection are difficult to distinguish from those of several other agents, especially Rickettsia rickettsii. Serologic testing is usually unable to provide evidence of CTF viral infection during the acute phase because of the late appearance of the various antibodies. Here we report the development and clinical application of a test to diagnose this disease during the acute stages. Oligonucleotide primers to the S2 segment of CTF (Florio) virus were made, and these were used in the amplification of a 528-bp fragment of DNA, transcribed from the double-stranded CTF virus RNA template by reverse transcriptase PCR. RNAs processed from 16 CTF virus isolates yielded similar results when analyzed on agarose gels. These were distinguishable from their antigenic relatives Eyach, S6-14-03, and T5-2092 and from other coltiviruses and an orbivirus but not from the antigenically distinct CTF virus-related isolate 720896. A mouse model demonstrated the utility of this method with whole-blood specimens, and CTF virus was successfully detected in human sera from the initial day of the onset of symptoms to 8 days later. The reverse transcriptase PCR method is a promising tool for the early diagnosis of CTF viral infection, or for ruling out CTF virus as the etiologic agent, in order to facilitate appropriate medical support.
Survey of emm gene sequences and T-antigen types from systemic Streptococcus pyogenes infection isolates collected in San Francisco, California; Atlanta, Georgia; and Connecticut in 1994 and 1995
Authors: Beall B , Facklam R , Hoenes T , Schwartz B
Published: January 2020

The variable 5' emm (M-protein gene) sequences and T-antigen types were determined from 340 systemic group A streptococcal (GAS) isolates taken from hospitalized patients in San Francisco, Calif.; Atlanta, Ga.; and Connecticut in 1994 and 1995. Eighty percent of these isolates had emm sequences and T-antigen types in agreement with previously recorded M- and T-antigen associations. Most of the remaining strains either were T nontypeable (11%) or contained emm genes encoding M proteins for which T-antigen associations have not been made (6%). One newly encountered emm gene, designated ST2974, from each of 13 isolates had the T type 8/25/Imp19. Another new emm gene, ST2967, from 8 of 11 isolates was T nontypeable. Six other unique emm gene sequences from seven isolates were encountered. Sequencing of the variable region of the emm gene of GAS isolates (emm typing) is effective for surveying the sequence variability of the M virulence protein, and combined with T typing, emm typing is useful for monitoring GAS strain diversity.
PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum
Authors: Miriam A , Griffiths S G , Lovely J E , Lynch W H
Published: January 2020

A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells.
Rapid identification of Candida species in blood cultures by a clinically useful PCR method
Authors: Shin J H , Nolte F S , Morrison C J
Published: January 2020

Widespread use of fluconazole for the prophylaxis and treatment of candidiasis has led to a reduction in the number of cases of candidemia caused by Candida albicans but has also resulted in the emergence of candidemias caused by innately fluconazole-resistant, non-C. albicans Candida species. Given the fulminant and rapidly fatal outcome of acute disseminated candidiasis, rapid identification of newly emerging Candida species in blood culture is critical for the implementation of appropriately targeted antifungal drug therapy. Therefore, we used a PCR-based assay to rapidly identify Candida species from positive blood culture bottles. This assay used fungus-specific, universal primers for DNA amplification and species-specific probes to identify C. albicans, C. krusei, C. parapsilosis, C. tropicalis, or C. glabrata amplicons. It also used a simpler and more rapid (1.5-h) sample preparation technique than those described previously and used detergent, heat, and mechanical breakage to recover Candida species DNA from blood cultures. A simple and rapid (3.5-h) enzyme immunosorbent assay (EIA)-based format was then used for amplicon detection. One hundred fifty blood culture bottles, including 73 positive blood culture bottle sets (aerobic and anaerobic) from 31 patients with candidemia, were tested. The combined PCR and EIA methods (PCR-EIA) correctly identified all Candida species in 73 blood culture bottle sets, including bottles containing bacteria coisolated with yeasts and 3 cultures of samples from patients with mixed candidemias originally identified as single-species infections by routine phenotypic identification methods. Species identification time was reduced from a mean of 3.5 days by routine phenotypic methods to 7 h by the PCR-EIA method. No false-positive results were obtained for patients with bacteremias (n = 18), artificially produced non-Candida fungemias (n = 3), or bottles with no growth (n = 20). Analytical sensitivity was 1 cell per 2-microl sample. This method is simpler and more rapid than previously described molecular identification methods, can identify all five of the most medically important Candida species, and has the potential to be automated for use in the clinical microbiology laboratory.
Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans
Authors: Pacheco A B , Guth B E , Soares K C , Nishimura L , de Almeida D F , Ferreira L C
Published: January 2020

The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.
Oligonucleotide (GTG)5 as an epidemiological tool in the study of nontuberculous mycobacteria
Authors: Cilliers F J , Warren R M , Hauman J H , Wiid I J , van Helden P D
Published: January 2020

Analysis of restriction fragment length polymorphisms in the genome of Mycobacterium tuberculosis (DNA fingerprinting) has proved to be a useful epidemiological tool in the study of tuberculosis within populations or communities. However, to date, no similar method has been developed to study the epidemiology of nontuberculous mycobacteria (NTM). In this communication, we report that a simple oligonucleotide repeat, (GTG)5, can be used to accurately genotype all species and strains of NTM tested. We suggest that this technology is an easily applied and accurate tool which can be used for the study of the epidemiology of NTM.
Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients
Authors: Woo P C , Chiu S S , Seto W H , Peiris M
Published: January 2020

Rapid diagnosis of respiratory viral infections in children resulted in significantly reduced hospital stays, antibiotic use, and laboratory utilization compared with those of a matched group of patients from the previous year who were diagnosed by virus culture. We demonstrate that rapid diagnosis of respiratory infections in children is a cost-effective procedure.
Detection of human Betaherpesvirinae in saliva and urine from immunocompromised and immunocompetent subjects
Authors: Gautheret-Dejean A , Aubin J T , Poirel L , Huraux J M , Nicolas J C , Rozenbaum W , Agut H
Published: January 2020

Human cytomegalovirus (HCMV) is a well-known opportunistic agent that reactivates in human immunodeficiency virus (HIV)-seropositive subjects. Human herpesvirus 6 (HHV-6) and HHV-7 were discovered recently and, like HCMV, belong to the Betaherpesvirinae subfamily. We looked for the presence of HCMV, HHV-6, and HHV-7 by PCR with saliva and urine samples from 125 HIV-seropositive patients at different stages of HIV infection and with saliva and urine samples from 29 HIV-seronegative subjects. All three viruses were frequently detected in the saliva (overall rates of detection, 61, 43, and 63% for HCMV, HHV-6, and HHV-7, respectively) with no correlation with the stage of immune deficiency. In contrast, HCMV was detected in urine much more frequently than the two other herpesviruses (overall rates of detection, 37, 2, and 6.5% for HCMV, HHV-6, and HHV-7, respectively) and was associated with immune deficiency. This suggests that these three genetically related viruses differ from each other with regard to replication in the urinary tract.
Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens
Authors: Mathis A , Weber R , Kuster H , Speich R
Published: January 2020

Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.
Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay
Authors: Pawlotsky J M , Prescott L , Simmonds P , Pellet C , Laurent-Puig P , Labonne C , Darthuy F , Remire J , Duval J , Buffet C , Etienne J P , Dhumeaux D , Dussaix E
Published: January 2020

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.
Interlaboratory agreement among results of human papillomavirus type 16 enzyme-linked immunosorbent assays
Authors: Strickler H D , Hildesheim A , Viscidi R P , Shah K V , Goebel B , Drummond J , Waters D , Sun Y , Hubbert N L , Wacholder S , Brinton L A , Han C L , Nasca P C , McClimens R , Turk K , Devairakkam V , Leitman S , Martin C , Schiller J T
Published: January 2020

Serological assays for measuring antibodies to human papillomavirus type 16 (HPV-16) virus-like particles (VLPs) have become important epidemiologic tools in recent years. However, the interlaboratory replicability of these assays has not been assessed. In this investigation, three laboratories tested a panel of specimens obtained from two different groups: 265 subjects in a vulvar cancer case-control study and 107 healthy volunteer blood donors. Each laboratory used an enzyme-linked immunosorbent assay (ELISA), but no attempt was made to standardize assay procedures among the three laboratories. The data showed good day-to-day intralaboratory replicability in laboratory 1 (correlation coefficient, > or = 0.88) and good intra-assay variability in laboratory 3 (correlation coefficient, > or = 0.93). Interlaboratory correlations, likewise, ranged between 0.61 and 0.80 in both case-control study subjects and healthy blood donors, indicating that ELISA optical density (OD) values between laboratories were linearly related regardless of the population. Kappa coefficients (kappa), based on each laboratory's categorical interpretation of its results (as positive or negative), showed good agreement (kappa, > 0.6) in case-control study subjects and moderate agreement (kappa, > or = 0.4) in blood donors, a population that had few strongly positive sera. When OD values near seropositive cutoffs were treated as indeterminates, there was little discordance between laboratories in either population. The data suggest that each laboratory measured the same humoral immune response and that their HPV-16 VLP ELISAs performed similarly (Pearson correlations). Interlaboratory differences, however, probably due to reagents and procedures, were considerably greater than intralaboratory day-to-day variability. Interlaboratory agreement in determining seropositivity (kappa) could be improved by sharing positive and negative serum controls and by treating marginal results as indeterminate. As part of continuing cooperation to improve interlaboratory agreement, we are preparing bulk serum control specimens to be shared and made available to interested researchers.
Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR
Authors: Pitkäranta A , Arruda E , Malmberg H , Hayden F G
Published: January 2020

Of 20 adults with acute community-acquired sinusitis (ACAS), rhinovirus was detected in specimens from 10 (50%) patients, including maxillary aspirates from 8 (40%) patients and nasal swabs from 9 (45%) patients, by reverse transcription-PCR (RT-PCR). Human coronavirus was detected by RT-PCR in nasal swabs from 3 of 20 patients but in no sinus secretions. These findings suggest that rhinovirus is an important cause of ACAS and that viral invasion of the sinus cavity itself may be a common event during the disease.
Characterization of a specific Mycobacterium paratuberculosis recombinant clone expressing 35,000-molecular-weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease
Authors: El-Zaatari F A , Naser S A , Graham D Y
Published: January 2020

Johne's disease is a chronic enteritis of ruminants associated with enormous worldwide economic losses for the dairy cow- and goat-rearing industries. Management limitations and eradication programs for this disease have been hampered by the lack of a simple and specific diagnostic test for the detection of subclinical cases. We used a recombinant clone expressing a 35,000-molecular-weight Mycobacterium paratuberculosis antigen (p35 antigen) from a previously constructed expression library of M. paratuberculosis in Escherichia coli. The DNA fragment encoding the p35 gene hybridized only to DNA from Mycobacterium avium complex, but not to DNAs from other mycobacteria and nonmycobacterial organisms. The seroreactivity of p35 was evaluated by immunoblotting against 57 reference serum samples obtained from infected and uninfected animals. p35 was recognized by sera from 100% of animals with advanced Johne's disease (clinical stage) (12 cattle, 2 goats, and 2 sheep) and by sera from 75% of 20 cattle with early infection (subclinical stage). None of the sera from 15 M. paratuberculosis-free cows, 3 Mycobacterium bovis BCG-infected tuberculous cattle, or 3 cows artificially inoculated with multiple doses of viable M. paratuberculosis reacted with p35. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 86, 100, 100, and 75%, respectively. The accuracy of p35 immunoblotting was superior to those of commercially available diagnostic tests for Johne's disease. These results suggest that the p35 recombinant protein has potential for use in the serodiagnosis of animals with Johne's disease at all stages of infection. The DNA fragment encoding p35 may also serve as a probe for identification of M. avium complex infection.
Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease)
Authors: Kordick D L , Hilyard E J , Hadfield T L , Wilson K H , Steigerwalt A G , Brenner D J , Breitschwerdt E B
Published: January 2020

Shortly after adopting a 6-week-old cat, a veterinarian was bitten on the left index finger. Within 3 weeks, he developed headache, fever, and left axillary lymphadenopathy. Initial blood cultures from the cat and veterinarian were sterile. Repeat cultures from the cat grew Bartonella-like organisms with lophotrichous flagella. Sera from the veterinarian were not reactive against Bartonella henselae, B. quintana, or B. elizabethae antigens but were seroreactive (reciprocal titer, 1,024) against the feline isolate. Sequential serum samples from the cat were reactive against antigens of B. henselae (titer, 1,024), B. quintana (titer, 128), and the feline isolate (titer, 2,048). Phenotypic and genotypic characterization of this and six additional feline isolates, including microscopic evaluation, biochemical analysis, 16S rRNA gene sequencing, DNA-DNA hybridization, and PCR-restriction fragment length polymorphism of the 16S gene, 16S-23S intergenic spacer region, and citrate synthase gene identified the isolates as B. clarridgeiae. This is the first report of cat scratch disease associated with B. clarridgeiae.
Comparison of Madin-Darby canine kidney cells (MDCK) with a green monkey continuous cell line (Vero) and human lung embryonated cells (MRC-5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture
Authors: Reina J , Fernandez-Baca V , Blanco I , Munar M
Published: January 2020

We report a comparative study of the MDCK, Vero, and MRC-5 cell lines in the isolation of the influenza A (IA) virus. We studied 746 samples in which 63 IA viruses were isolated. The MDCK line displayed 100% sensitivity, the Vero line displayed 71.4%, and the MRC-5 displayed 57.1%. The MDCK line showed a statistically significant difference with respect to the Vero line (P = 0.001) and the MRC-5 line (P = 0.001). The quantitative sensitivity analysis showed the MDCK line to be superior to the other lines. It seems that the MDCK line is still one of the most recommendable for the isolation of the IA virus from respiratory samples.
Correlation of in vitro susceptibility results for amoxicillin-clavulanate and ampicillin-sulbactam tested against Escherichia coli
Authors: O'Shaughnessy E M , Fahle G A , Witebsky F G
Published: January 2020

The results of amoxicillin-clavulanate (AUG) and ampicillin-sulbactam (A/S) susceptibility testing by three different susceptibility testing methods, the MicroScan, Etest, and Kirby-Bauer methods, for 61 consecutive isolates of ampicillin-resistant Escherichia coli from different patients were compared. There was poor correlation of results for the two agents, the most and least marked discrepancies being observed by the MicroScan method (86.9% susceptible to AUG and 4.9% susceptible to A/S) and the Kirby-Bauer method (39.4% susceptible to AUG and 32.8% susceptible to A/S), respectively. More organisms were susceptible to AUG than A/S, regardless of the susceptibility testing methodology. The results from a College of American Pathologists survey with one E. coli isolate tested at different institutions also indicated greater susceptibility to AUG than to A/S. These agents are thought to be equally efficacious clinically. The discrepancies observed among methods for each antimicrobial inhibitor combination and the discrepancies observed between the two agents by each testing method suggest that the breakpoints for these agents need to be reevaluated.
Improved diagnosis of primary Toxoplasma gondii infection in early pregnancy by determination of antitoxoplasma immunoglobulin G avidity
Authors: Jenum P A , Stray-Pedersen B , Gundersen A G
Published: January 2020

The ability to discriminate between primary Toxoplasma gondii infection acquired in early pregnancy and infection that occurred prior to pregnancy was assessed by an enzyme immunoassay (EIA) to determine the avidity of toxoplasma-specific immunoglobulin G (IgG). The results were compared to those of the Platelia Toxo-IgM EIA and the dye test. The mean IgG avidity of 73 serum samples collected within 20 weeks after the estimated time of infection was 5.9%. Among 26 serum samples showing latent infection (toxoplasma-specific IgG positive and IgM negative) and 56 IgM-positive serum samples with a low dye test titer (<300 IU/ml), the mean avidities were 51.3 and 57.5%, respectively. A total of 72.8% of 92 IgM-positive serum samples with a high dye test titer (>300 IU/ml), suggesting a recent toxoplasma infection, had an IgG avidity of >20%, indicating that the infection started more than 20 weeks earlier. By introducing high IgG avidity as a criterion in the first half of pregnancy to exclude the possibility that toxoplasma infection was acquired during gestation, many women will avoid unnecessary examinations, treatment, and anxiety.
Phylogenetic placement and characterization of a new alpha-2 proteobacterium isolated from a patient with sepsis
Authors: Blomqvist G , Wesslén L , Påhlson C , Hjelm E , Pettersson B , Nikkilä T , Allard U , Svensson O , Uhlén M , Morein B , Friman G
Published: January 2020

An alpha-2 proteobacterium, previously unknown as determined by its phylogenetic characteristics and the DNA sequence of its 16S rRNA gene, was isolated from a patient who presented an unusual clinical picture, including high remitting fever and multiorgan involvement. The bacterium was detected in multiple plasma samples, obtained during the acute phase of the disease, after cocultivation in cell culture media. Electron microscopy of the organism showed a three-layer laminar cell wall and electron-dense granules within the cytoplasm, as well as a polar flagellum. By means of PCR followed by sequencing of amplified 16S ribosomal DNA fragments, the bacterium was found to differ from all species for which ribosomal sequence information is available. It is here provisionally named the Rasbo bacterium. At a subsequent relapse, the bacterium was identified in pericardial fluid both by PCR/sequencing and by direct electron microscopy. At a second relapse, it was again cultured from plasma. After in vitro adaptation to solid media, the MICs of various antibiotics could be determined. A transient immunoglobulin M (IgM) but no IgG response to the bacterium was found by an indirect immunofluorescence test, as well as by an immobilization test during the acute phase of the disease.
Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates
Authors: Steffan P , Vazquez J A , Boikov D , Xu C , Sobel J D , Akins R A
Published: January 2020

We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species. DNA from individual colonies can be amplified from crude single-colony lysates. Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species. RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time. Most (91%) of these identifications agreed with those assigned by API 20C tests. In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct. This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools. The identities of approximately 30% of C. albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting. Data suggest that clinical isolates conventionally identified as C. albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts. RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents in the clinical setting.
Diagnosis of genitourinary Chlamydia trachomatis infections by using the ligase chain reaction on patient-obtained vaginal swabs
Authors: Hook E W , Smith K , Mullen C , Stephens J , Rinehardt L , Pate M S , Lee H H
Published: January 2020

We compared the ligase chain reaction (LCR) assay to cell culture for diagnosis of genitourinary chlamydial infections in women using swab specimens obtained by clinicians from the endocervix and by patients from their own vaginas. Specimens from 40 (12.9%) of 309 patients were positive for chlamydial infection by culture, while the specimens of 50 (16.2%) patients were positive by LCR. Chlamydia trachomatis infection was verified for 9 of 10 patients whose LCR specimens were positive but whose cultures were negative. Vaginal and cervical swab specimens were positive by LCR for 46 (93.9%) and 44 (89.8%) of 49 chlamydia-infected patients, respectively. These data suggest that LCR testing for chlamydia with vaginal swab specimens obtained by patients themselves is as sensitive as cervical LCR and more sensitive than cell culture.
Frequency of isolation of Staphylococcus intermedius from humans
Authors: Mahoudeau I , Delabranche X , Prevost G , Monteil H , Piemont Y
Published: January 2020

We collected 3,397 consecutive isolates of coagulase-positive staphylococci from various specimens of hospitalized patients. All were retrospectively classified as Staphylococcus aureus, except two which were identified as S. intermedius: one isolated from the nasal flora of a healthy carrier and the other isolated from pleural fluid, probably as a sample contaminant.
Physical Assay and Growth Cycle Studies of a Defective Adeno-Satellite Virus
Authors: Parks Wade P. , Melnick Joseph L. , Rongey Robert , Mayor Heather D.
Published: January 2020

Electron microscopic particle counting of the defective adeno-satellite virus (ASV), by use of pseudoreplication and negative staining with phosphotungstic acid, was shown to be a reproducible quantitative assay procedure. Particles of satellite type 4 that were counted in fluids from infected cultures had the same morphology as particles that banded at a buoyant density of 1.43 g/cc in cesium chloride. Other satellite virus serotypes examined in the same manner had a buoyant density of 1.37 to 1.38 g/cc. A comparison of satellite titers obtained by complement fixation and by particle counting demonstrated that an increase in satellite particles resulted in a corresponding increase in CF titers; however, electron microscopy was at least 10 times more sensitive than complement fixation for detecting satellite virus. Growth cycle studies of satellite virus in cells co-infected with adenovirus, as assayed by particle counting, indicated that the kinetics of satellite virus production closely followed the kinetics of its helper adenovirus production, with an eclipse period of 12 to 16 hr. The eclipse period of the satellite remained the same when cultures were preinfected with satellite 24 hr prior to adenovirus inoculation. However, when cultures were infected with adenovirus 12 hr before satellite virus, the eclipse period of the satellite was shortened to between 4 and 6 hr. Thus, satellite virus replication seems dependent upon a relatively late event in the adenovirus replication cycle. When cells were co-infected with adenovirus and its defective satellite, the yield of adenovirus was markedly reduced from that obtained in cells singly infected with adenovirus.
Distribution of Cell Receptors and Simple Sugar Inhibitors During Encephalomyocarditis Virus-Cell Union
Authors: Kunin Calvin M.
Published: January 2020

Encephalomyocarditis (EMC) virus was studied for ability to agglutinate erythrocytes of various species. Human, rat, and guinea pig erythrocytes, as well as those from young rabbits, were readily agglutinated. Cells from older rabbits absorbed virus poorly, and showed little agglutination. Uptake of virus by rabbit brain also diminished with age. Various mouse tissues absorbed virus about equally well. Hemagglutination-inhibition studies demonstrated that a number of simple sugars, particularly glucose and galactosamine, interfered with uptake of virus by cells. Dextran sulfates were highly active inhibitors of EMC hemagglutination. Attempts to localize the site of action of the sugars on virus or cell are described. Treatment of virus with periodate or
p
-chloromercurobenzoate, and acetylation of virus, inhibited hemagglutination, but acetylation of semipurified receptor did not. Clarification of the nature of the virus-cell union will require studies to identify possible specific sugars in the virus capsid and the cell receptor.
Effect of Bacteriophage R17 Infection on Hostdirected Synthesis of Ribosomal Ribonucleates
Authors: Hudson James B. , Paranchych William
Published: January 2020

Studies were performed on the synthesis of ribosomal ribonucleates in cells of
Escherichia coli
K-12 infected by the ribonucleic acid (RNA) bacteriophage R17. Host-specific RNA was measured in the presence of phage RNA by in vitro hybridization of the purified ribonucleates with
E. coli
deoxyribonucleic acid. The results showed that, although the overall rate of RNA synthesis was only slightly affected by phage infection, the level of host RNA synthesis was decreased by 70 to 80%. Fractionation of the purified ribonucleates by sucrose gradient sedimentation, followed by hybridization of fractions sedimenting in the 23
S
and 16
S
regions, revealed that the level of ribosomal RNA synthesis was also decreased by 70 to 80%, and that this inhibition occurred during the first 15 to 20 min after infection. These findings are discussed in light of what is known about the inhibition of host RNA synthesis by other virus systems.
Purification and Chemistry of Bacteriophage ϰ
Authors: Schade Sylvia Z. , Adler Julius
Published: January 2020

From a stock of ϰ phage grown on
Salmonella
, a host-range mutant which attacks
Escherichia coli
was isolated. As in the case of
Salmonella
, only motile strains of
E. coli
are sensitive to ϰ. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 × 10
−9
ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage ϰ was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.
Structural Units of Reovirus Ribonucleic Acid and Their Possible Functional Significance
Authors: Watanabe Y. , Graham A. F.
Published: January 2020

Reovirus contains ribonucleic acid (RNA) equivalent in amount to a molecular weight of approximately 10
7
daltons. On isolation, this RNA is invariably broken into fragments of three different sizes. The three pieces have been separated from each other by chromatography on methylated albumin-kieselguhr columns. Denaturation of the three fragments of RNA in dimethyl sulfoxide led to separation of the strands, as suggested by sucrose gradient sedimentation patterns and by the second-order kinetics of reannealing. Molecular weights of 0.8 × 10
6
, 1.4 × 10
6
, and 2.4 × 10
6
were determined for the double-stranded fragments from the sedimentation rates of the single-stranded RNA obtained by denaturation. There was little or no homology among the three classes of denatured RNA when taken in pairs in hybridization tests. The three pieces of double-stranded RNA, therefore, did not result from random breaks in the original viral RNA molecule. Virusspecific single-stranded RNA formed in infected cells, and previously found to be largely if not entirely messenger RNA transcribed from the viral genome, was also separated into three size classes by sedimentation through sucrose gradients. Each class of single-stranded RNA corresponded in size to one of the three fragments of double-stranded RNA. The largest piece of single-stranded RNA hybridized uniquely with the largest fragment of denatured viral RNA. By whatever means this fragment of double-stranded RNA may be joined into the viral RNA molecule, it seems to act as a specific unit for transcription of an uninterrupted messenger RNA of equivalent length.
Morphogenesis of Avian Infectious Bronchitis Virus and a Related Human Virus (Strain 229E)
Authors: Becker Walter B. , McIntosh Kenneth , Dees Jane H. , Chanock Robert M.
Published: January 2020

Avian infectious bronchitis virus (IBV) and strain 229E, a virus recently recovered from patients with colds, have been shown to possess a similar distinctive morphology in negatively stained preparations. An electron microscopic study of the morphogenesis of IBV in the chorioallantoic membrane and of strain 229E in WI-38 cells was performed. In infected cells, round electron-dense particles 82 mμ in diameter were observed to form by a process of budding from membranes of the endoplasmic reticulum and cytoplasmic vesicles. The particles in IBV-infected cells were similar in size and shape to those in strain 229E-infected cells but showed certain differences in internal structure. The evidence that the particles represent virions and the implications of these findings in the classification of this virus group are discussed.
Electron Microscopy of the Formation of Wound Tumor Virus in Abdominally Inoculated Insect Vectors
Authors: Shikata Eishiro , Maramorosch Karl
Published: January 2020

Fifth-instar nymphs of
Agallia constricta
leafhoppers were injected abdominally with extracts from root tumors confining wound tumor virus (WTV). The insects were sacrificed at predetermined intervals, their internal organs dissected, fixed, embedded, sectioned, stained, and examined in a Siemens Elmiskop I. Sequential stages in virus development were reconstructed from consecutive samples of fatbody tissues. Changes resulting from the infection were: (i) a viroplasm, i.e., an accumulation of electron-dense aggregates; (ii) the appearance at the periphery of the viroplasm of a few fully formed virus particles recognized as virions; (iii) the formation of increasing numbers of individual virions, not only at the periphery but also in the viroplasm; (iv) the engulfing of virions within multimembranous structures; and (v) the formation of virus microcrystals either at the sites of former viroplasms, or at some distance. These morphological findings indicate that, following abdominal inoculation of WTV, the plant-pathogenic virus develops within the cytoplasmic matrix proper of insect vector cells. In addition to the fatbody tissues, WTV was detected in the epidermis, muscles, and trachea of abdominally inoculated insects, demonstrating the systemic invasion of the mechanically infected arthropod host. No virus was found in the gut tissues.
Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells
Authors: Carp Richard I.
Published: January 2020

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.
Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid
Authors: Friedman Robert M. , Fantes Karl H. , Levy Hilton B. , Carter William B.
Published: January 2020

Actinomycin D-treated chick fibroblasts were infected with purified
32
P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42
S
, 30
S
, and 16
S
were present. The 42
S
form corresponded to the RNA of the virion. The 16
S
form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 μg/ml) or cycloheximide (200 μg/ml) added at the time of virus infection, had no effect on the formation of the 30
S
RNA but inhibited the production of the 16
S
form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of
32
P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16
S
RNA. No protein synthesis appears necessary for formation of 30
S
RNA from parental virus RNA.
In Vitro and In Vivo Observations on a Murine C-Type Virus
Authors: Hall William T. , Andresen William F. , Evans Virginia J.
Published: January 2020

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.
Vaccinia Virus Infection of Synchronized Pig Kidney Cells
Authors: Groyon Rose Mary , Kniazeff Alexis J.
Published: January 2020

Vaccinia virus replication was studied in pig kidney (PK-15) cells synchronized by excess thymidine treatment. It was found that virus replication with concomitant inhibition of mitosis can occur at any period in the life cycle of the cell except for the narrow span of time from late prophase through telophase. Cells infected at this time continue to divide, and vaccinia does not replicate until cell division is complete.