bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test
Authors: Boland G J , de Weger R A , Tilanus M G , Ververs C , Bosboom-Kalsbeek K , de Gast G C
Published: January 2020

To compare the sensitivity and suitability of detection of active cytomegalovirus (CMV) infection by using monoclonal antibodies against CMV antigen (antigen test to detect antigenemia) and the polymerase chain reaction (PCR; to detect viral DNA) in granulocytes, 19 heart and 2 lung transplant recipients were closely monitored by these tests for at least 3 months after transplantation. All patients were CMV seropositive or had a seropositive donor. In total, 201 samples were tested; 46 were positive by both tests, 9 samples showed only antigenemia, 54 samples were positive by PCR only, and 102 samples were negative by both tests. PCR was positive earlier after transplantation in eight patients, whereas antigenemia was positive earlier after transplantation in one patient. In another four patients, both tests were positive at the same time. PCR was, on average, positive for a longer period of time. Discordant results showing a positive antigen test and a negative PCR were partly due to sampling error; some were positive by PCR on retesting. Samples which were negative by the antigen test and positive by PCR were taken at the beginning or at the end of an active CMV infection. In two patients, no active CMV infection was detected by the antigen test, cultures of urine and saliva, or serology, although PCR was positive for a long period of time in the two patients.
Human immunodeficiency virus type 1 is present in the cerebrospinal fluid of a majority of infected individuals
Authors: Chiodi F , Keys B , Albert J , Hagberg L , Lundeberg J , Uhlén M , Fenyö E M , Norkrans G
Published: January 2020

Cerebrospinal fluid (CSF) specimens from 63 patients with different severities of human immunodeficiency virus (HIV-1) infection, including asymptomatic virus carriers, were examined for the presence of HIV-1 by using polymerase chain reaction (PCR) and virus isolation. Polyadenylated RNA, presumably associated with virus particles, was extracted and reverse transcribed, and the pol region was amplified in a nested PCR. Virus could be detected in 90% of the CSF specimens examined by PCR, and data on isolation of virus from CSF were in agreement with these figures. In fact, when several CSF specimens from the same individual were studied, HIV-1 could be isolated from 80% of the patients. The presence of the viral RNA in CSF was independent of the clinical stage of infection and of neurological symptoms. These results show that the spread of HIV-1 to the brain represents an early event during infection and occurs in the majority of asymptomatic individuals.
Evaluation of the bead enzyme-linked immunosorbent assay for detection of cholera toxin directly from stool specimens
Authors: Ramamurthy T , Bhattacharya S K , Uesaka Y , Horigome K , Paul M , Sen D , Pal S C , Takeda T , Takeda Y , Nair G B
Published: January 2020

A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically, and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae O1 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. cholerae O1 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/ml and greater than 100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories.
Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group. The NIH/NIAID/DAIDS/ACTG Virology Laboratories
Authors: Hollinger F B , Bremer J W , Myers L E , Gold J W , McQuay L
Published: January 2020

An independent quality assurance program has been established by the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring virologic assays performed by nearly 40 laboratories participating in multicenter clinical trials in the United States. Since virologic endpoints are important in evaluating the timing and efficacy of therapeutic interventions, it is imperative that virologic measurements be accurate and uniform. When the quality assurance program was initially created, fewer than 40% of the laboratories could consistently recover human immunodeficiency virus (HIV) from peripheral blood mononuclear cells (PBMCs) of HIV-infected patients. By comparing coculture procedures in the more competent laboratories with those in laboratories who were struggling to isolate virus, optimal conditions were established and nonessential reagents and practices were eliminated. Changes were rapidly introduced into a laboratory when experience dictated that such modifications would result in a favorable outcome. Isolation of HIV was enhanced by optimizing the numbers and ratios of patient and donor cells used in cultures, by standardizing PBMC separation procedures, by using fresh rather than frozen donor PBMCs, by processing whole blood within 24 h, and by using natural delectinated interleukin 2 instead of recombinant interleukin 2 products in existence at that time. Delays of more than 8 h in the addition of phytohemagglutinin-stimulated donor cells to freshly separated patient PBMCs reduced recovery. Phytohemagglutinin in cocultures and the addition of Polybrene and anti-human alpha interferon to media were not important in HIV isolation. The introduction of a consensus protocol based on this information brought most laboratories quickly into compliance. In addition, monthly monitoring has successfully maintained proficiency among the laboratories, a process that is critical for the scientific integrity of collaborative multicenter trials. Problems which might not be appreciated for months are now being resolved early, before data can be compromised unknowingly. This consensus protocol is recommended for any laboratory attempting to isolate HIV for the purpose of standardizing recovery and for accessing virologic endpoints in clinical trials.
Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes
Authors: Coffin J W , Condon C , Compston C A , Potter K N , Lamontagne L R , Shafiq J , Kunimoto D Y
Published: January 2020

Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.
Comparison of the VIDAS Clostridium difficile toxin A immunoassay with C. difficile culture and cytotoxin and latex tests
Authors: Shanholtzer C J , Willard K E , Holter J J , Olson M M , Gerding D N , Peterson L R
Published: January 2020

The VIDAS Clostridium difficile toxin A immunoassay (CDA) is a new, automated, enzyme-linked fluorescent-antibody assay for detection of C. difficile toxin A antigen in stool specimens. Simultaneous, parallel testing was performed by using the VIDAS CDA, the Culturette brand CDT latex test for C. difficile antigens, and conventional laboratory cell culture tests for C. difficile, cytotoxicity and C. difficile culture. One hundred ninety-four consecutive fresh soft or liquid stool samples submitted for C. difficile testing between July and September 1990 were evaluated. Of the 194 samples tested, 19 (10%) were from 16 patients who met our case definition for C. difficile-associated disease. The in vitro tests were evaluated in relation to two forms of a clinical case definition. In one form, a positive culture for toxin-producing C. difficile or a positive cytotoxin result obtained directly from the stool specimen was required as laboratory evidence of C. difficile. In the other, a positive result of any of the four laboratory tests was accepted for the laboratory portion of the case definition. No significant difference between the sensitivity of the VIDAS CDA and that of the Culturette brand CDT latex test was found (48 to 58% sensitivity for the CDT latex test and 52 to 63% sensitivity for the VIDAS CDA compared with 93 to 100% sensitivity for culture and 70 to 100% sensitivity for cytotoxin testing). The performance of the VIDAS CDA, however, was hampered by a high percentage of tests (19%) which gave an uninterpretable result.
Characterization of rubella virus-specific antibody responses by using a new synthetic peptide-based enzyme-linked immunosorbent assay
Authors: Mitchell L A , Zhang T , Ho M , Décarie D , Tingle A J , Zrein M , Lacroix M
Published: January 2020

Rubella virus (RV)-specific immunoglobulin G antibodies were studied by enzyme-linked immunosorbent assay (ELISA) techniques in sera from RV (RA 27/3)-vaccinated individuals, patients experiencing natural RV infection, congenital rubella syndrome patients, and individuals failing to respond to repeated RV immunization. Results obtained by using whole-RV ELISAs (detergent-solubilized M33 strain or intact Gilchrist strain) and hemagglutination inhibition (HAI) and neutralization (NT) assays were compared with results obtained with the same sera by using ELISAs employing a synthetic peptide, BCH-178, representing a putative neutralization domain on the RV E1 protein. Murine RV E1-specific monoclonal antibodies with HAI and NT activities exhibited strong reactivity in ELISAs with BCH-178 peptide. In sera from RA 27/3-vaccinated individuals collected at 0 (prevaccine), 1, 2, 3, 4, 5, 6, 12, and 24 to 52 weeks postvaccine, the development of E1-peptide-reactive antibodies closely paralleled increases in RV-specific antibodies measured by whole-RV ELISAs and HAI and NT assays. Similarly, sequential serum samples obtained from patients during acute and convalescent phases of natural RV infection showed a coordinate increase in RV-specific antibodies as measured by whole-RV and peptide ELISAs. Conversely, congenital rubella syndrome patient sera, although exhibiting high levels of antibody in whole-RV ELISAs, had little or no antibody directed to the neutralization domain peptide. Sera from patients failing to respond to repeated RV immunization contained very low levels of RV-specific antibody in all ELISAs. Our results that the sequence represented by BCH-178 peptide may be a previously unidentified neutralization epitope for human antibodies on the RV E1 protein and may prove useful in determining effective RV immunity.
Distribution of heartwater in the Caribbean determined on the basis of detection of antibodies to the conserved 32-kilodalton protein of Cowdria ruminantium
Authors: Muller Kubold A , Martinez D , Camus E , Jongejan F
Published: January 2020

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect immunoglobulin G antibodies to the major 32-kDa protein of Cowdria ruminantium. A total of 1,804 serum samples collected from cattle on 19 islands in the eastern Caribbean Basin were tested by this cELISA. A total of 133 serum samples from 10 islands (Antigua, Dominica, Grenada, Guadeloupe, Martinique, Montserrat, St. Kitts, St. Lucia, St. Martin, and St. Vincent) were found to be positive. The presence of antibodies to C. ruminantium in cattle on these islands was confirmed by immunofluorescence and Western blotting (immunoblotting). In earlier studies, C. ruminantium has been demonstrated only on Guadeloupe, Antigua, and Marie Galante. This study shows that the causative agent of heartwater is now firmly established in the Caribbean.
Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica
Authors: Ibrahim A , Liesack W , Stackebrandt E
Published: January 2020

The polymerase chain reaction technique was used to develop a rapid diagnostic assay for detection of pathogenic Yersinia enterocolitica strains. The assay targeted a stretch of 163 bp of the yst gene and could be applied to both pure cultures and crude DNA extracted from feces. The defined primer pair amplified the targeted sequence from only pathogenic strains and fecal samples seeded with the serotype O:3 strain of Y. enterocolitica, whereas neither nonpathogenic strains nor normal stools yielded any amplified fragments. Of the other Yersinia species and non-Yersinia species tested, only two strains of Y. kristensenii yielded the same amplified product. A 20-mer oligonucleotide probe specifically hybridized within the amplified yst fragment of Y. enterocolitica but did not hybridize with the amplified yst fragment of Y. kristensenii by Southern and dot blot hybridizations. This confirms the reliability of this diagnostic assay in both clinical and epidemiological studies. The availability of the extracted DNA for the polymerase chain reaction was checked by simultaneous amplification of a part of the 16S rDNA and the yst gene. The entire diagnostic assay, including a simplified technique for DNA extraction, the amplification process, and gel electrophoresis, could be completed within 1 working day, which is better than the time required for the time-consuming traditional techniques used in clinical laboratories.
Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae
Authors: Roblin P M , Dumornay W , Hammerschlag M R
Published: January 2020

Chlamydia pneumoniae has proved to be difficult to isolate and propagate in cell culture. We compared the growth of three strains of C. pneumoniae, TW-183 and two clinical isolates from Brooklyn, N.Y., in five cell lines, including HeLa 229, McCoy, HL, HEp-2, and HTED, an immortalized human tracheal cell line. HEp-2 was the most sensitive cell line tested. When 10-fold dilutions of three C. pneumoniae strains at known titers were inoculated into the different cell lines, the mean number of inclusion-forming units per milliliter was 1 to 2 log units higher in the HEp-2 than in the other cell lines. This difference was statistically significant. Omission of pretreatment with DEAE-dextran resulted in larger inclusions than those seen in pretreated cells, with the exception of McCoy and HTED cells. Retrieval of clinical specimens previously cultured on HeLa 229 cells and comparison of mean inclusion counts in fresh clinical specimens simultaneously inoculated on HeLa 229 and HEp-2 cells suggested that culture in HEp-2 cells may require only the initial inoculation and one passage, compared with three to four passages, as required by culture in HeLa 229 cells.
Immunodominant regions within the hepatitis C virus core and putative matrix proteins
Authors: Sällberg M , Rudén U , Wahren B , Magnius L O
Published: January 2020

The complete amino acid sequences of hepatitis C virus (HCV) core (residues 1 to 115) and putative matrix (residues 116 to 190) proteins were synthesized as 18-residue-long peptides with an 8-amino-acid overlap. The peptides were assayed with 50 human serum samples with antibodies to HCV (anti-HCV) and 46 serum samples without anti-HCV, as determined by several commercial assays. Immunodominant regions were defined within residues 1 to 18, 11 to 28, 21 to 38, 51 to 68, and 101 to 118. The peptides that covered these regions were recognized by 40 of 50 (80%), 42 of 50 (84%), 36 of 50 (72%), 34 of 48 (68%), and 36 of 48 (72%) of the anti-HCV positive serum samples, respectively. Two anti-HCV negative serum samples were each repeatedly reactive with one peptide, but both were found to be negative by confirmatory anti-HCV assays. Four serum samples that were confirmed to be positive for anti-HCV in commercial assays did not recognize any of the peptides that cover the HCV core-matrix regions. Ninety-two percent of anti-HCV-positive serum samples reacted with a combination of peptides covering residues 1 to 18 and 11 to 28. Testing of peptides that contain the reported genotypic variations of the HCV core within the regions at residues 1 to 18, 51 to 68, and 101 to 118 showed that a change from Thr-110 to Asn-110 decreased the reactivities of eight serum samples. In conclusion, we found that human antibodies to the HCV core-matrix protein(s) are mainly directed to linear determinants and can easily be reproduced by using short synthetic peptides. We also found that such antibodies develop in more than 90% of HCV-infected people.
Application of ubiquinone systems and electrophoretic comparison of enzymes to identification of clinical isolates of Aspergillus fumigatus and several other species of Aspergillus
Authors: Matsuda H , Kohno S , Maesaki S , Yamada H , Koga H , Tamura M , Kuraishi H , Sugiyama J
Published: January 2020

The ubiquinone systems and electrophoretic comparison of enzymes were used to determine the relatedness among 64 isolates of seven Aspergillus spp. These were 31 clinical and 3 nonclinical isolates of Aspergillus fumigatus Fres., 2 isolates of A. nidulellus Samson & W. Gams, 8 isolates of A. terreus Thom, 4 isolates of A. flavus Link, 1 isolate of A. oryzae (Ahlburg) Cohn, 14 isolates of A. niger van Tieghem, and 1 isolate of A. japonicus Saito. The enzymes glucose 6-phosphate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, fumarase, and malate dehydrogenase were examined. The relative mobilities were analyzed numerically. The results were presented as a dendrogram. Isolates from clinical and nonclinical sources within the same species had identical ubiquinone systems and identical or very similar enzyme patterns. In the dendrogram, 64 of the tested isolates were separated into seven major clusters at a 60% similarity level. Each major cluster corresponds to a single species. On the dendrogram, A. fumigatus isolates showed homogeneity, whereas A. niger isolates showed relative heterogeneity; in particular, A. niger MF-24 and the other A. niger isolates were distantly linked to each other. All A. fumigatus isolates had the Q-10 ubiquinone system and formed a single major cluster at a similarity level of 73% or greater. Glucose 6-phosphate dehydrogenase and glutamate dehydrogenase were key enzymes for differentiating all clinical and nonclinical isolates of A. fumigatus from the other Aspergillus spp. Ubiquinone systems and enzyme patterns appear to be objective and useful indicators for use in the precise identification of clinical isolates of Aspergillus spp.
Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays
Authors: Paulsson M , Ljungh A , Wadström T
Published: January 2020

Seventeen strains of ten different species of coagulase-negative staphylococci were shown to interact with collagen, laminin, fibronectin, and vitronectin immobilized on latex beads. Different species of coagulase-negative staphylococci have different capacities to agglutinate proteins. Cells of 18 strains of Staphylococcus haemolyticus reacted more strongly than did cells of 18 Staphylococcus epidermidis strains with proteincoated latex beads, although no significant difference in cell surface hydrophobicity or charge could be shown. The cell surface receptors of S. haemolyticus were more heat and protease resistant than were Staphylococcus aureus receptors. Strains of Staphylococcus saprophyticus isolated from urinary tract infections showed a high capacity to adhere to laminin. The ability to agglutinate fibronectin and collagen was common among coagulase-negative staphylococci isolated from other infections; 55% (31 of 56) and 63% (35 of 56) agglutinated fibronectin and/or collagen. S. haemolyticus and S. epidermidis bound to both N-terminal (29-kDa) and C-terminal (120-kDa) fragments of fibronectin.
Standardization of an enzyme immunoassay for human antibody to Haemophilus ducreyi
Authors: Desjardins M , Thompson C E , Filion L G , Ndinya-Achola J O , Plummer F A , Ronald A R , Piot P , Cameron D W
Published: January 2020

We standardized a serologic enzyme immunoassay (EIA) for human immunoglobulin G and M antibodies against Haemophilus ducreyi. We evaluated the performance of this test with respect to the time from acute chancroid and coinfection with human immunodeficiency virus (HIV). Antibody to a crude, soluble bacterial antigen of one H. ducreyi strain was detected in a panel of serum samples from clinically and microbiologically confirmed cases of chancroid and from controls. Test interpretation was standardized for optimal sensitivity and specificity. Performance of the EIA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection. The EIA performed adequately as a serologic screening test for field evaluation and epidemiologic application in conjunction with sexually transmitted disease and HIV detection and control efforts.
Detection of salivary antibodies in cats infected with feline immunodeficiency virus
Authors: Poli A , Giannelli C , Pistello M , Zaccaro L , Pieracci D , Bendinelli M , Malvaldi G
Published: January 2020

The saliva of cats infected with feline immunodeficiency virus was examined for total immunoglobulin content and antiviral antibodies. Seropositive cats showed an increase in salivary immunoglobulin G levels, which was only partly attributable to the enhanced prevalence of oral inflammatory lesions, compared with the levels in seronegative cats. Immunoglobulin G, but not immunoglobulin M, levels in serum were also increased. Salivary antibodies were determined by indirect immunofluorescence and Western blot (immunoblot) analysis. All but 1 of the 16 seropositive cats examined were positive, while all 16 control cats were negative. The presence of oral lesions was not a prerequisite for antibody detection in saliva. It was concluded that salivary antibody might be usefully exploited for diagnostic and epidemiologic purposes.
Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination
Authors: Musser J M , Kapur V
Published: January 2020

Genetic relationships among 254 isolates of Staphylococcus aureus resistant to methicillin recovered between 1961 and 1992 from nine countries on four continents were determined by analyzing electrophoretically demonstrable allelic variation at 15 chromosomal enzyme loci. Fifteen distinctive electrophoretic types, marking clones, were identified. The mec gene is harbored by many divergent phylogenetic lineages representing a large portion of the breadth of chromosomal diversity in the species, a result that is interpreted as evidence that multiple episodes of horizontal transfer and recombination have contributed to the spread of this resistance determinant in natural populations. Isolates recovered in the United Kingdom, Denmark, Switzerland, Egypt, and Uganda in the 1960s are of a single multilocus enzyme genotype and probably are progeny of an ancestral methicillin-resistant clone. There is geographic variation in the frequency of recovery of the common methicillin-resistant clones, an observation that may in part explain reported regional differences in natural history correlates of resistant organisms.
Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis
Authors: White-Owen C , Alexander J W , Sramkoski R M , Babcock G F
Published: January 2020

A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by human neutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 microliters of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commercially available whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external free or adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalized bacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size, (ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability to distinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes as quenching agents, and (vi) ability to fix cells and store for future FCM analysis.
Excretory-secretory antigenic components of Paragonimus heterotremus recognized by infected human sera
Authors: Maleewong W , Wongkham C , Intapan P , Pariyanonda S , Morakote N
Published: January 2020

Antigenic components of Paragonimus heterotremus metabolic products were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis of sera from patients with P. heterotremus infection, from patients with other illnesses, and from healthy adults. By SDS-PAGE, it was found that the metabolic products comprised more than eight major polypeptides. Immunoblot analysis revealed 11 components which were strongly recognized by paragonimiasis antisera. These antigenic components had molecular masses ranging from less than 12.3 kDa to 144 kDa. One antigenic band of 31.5 kDa was found to give a consistent reaction with paragonimiasis antisera (97% sensitivity). Of the other patient sera, only sera from patients with Fasciola sp. infection reacted with antigenic bands of 56, 38, and 18.5 kDa. The present findings suggest that the 31.5-kDa component is sensitive and specific for the diagnosis of human P. heterotremus paragonimiasis.
Detection of genes in feces by booster polymerase chain reaction
Authors: Saulnier P , Andremont A
Published: January 2020

A 321-bp fragment intragenic to the gene ereA carried by Escherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.
Ribotyping provides efficient differentiation of nosocomial Serratia marcescens isolates in a pediatric hospital
Authors: Bingen E H , Mariani-Kurkdjian P , Lambert-Zechovsky N Y , Desjardins P , Denamur E , Aujard Y , Vilmer E , Elion J
Published: January 2020

Ribotyping with a nonradioactive probing system was used for the epidemiological evaluation of 15 Serratia marcescens nosocomial strains isolated from the stools of 12 children with no apparent illness in five different hospital wards over a 20-day period. Our results indicate that the occurrence of S. marcescens colonization was the result of the spread of a single epidemiological strain in the hematology ward, the oncology ward, and the gastroenterology ward and in two neonates in the neonatology ward, suggesting cross-contamination between the patients in these four wards. This isolate was genotypically unrelated to the bacterial strain found in the three other patients in the neonatology ward. Interestingly, one patient in the neonatology ward harbored these two genotypically different strains. Finally, the patient in the intensive care unit was colonized with a different strain. We find ribotyping to be a more reliable technique than biochemical typing. The results of ribotyping are more easily interpreted than are those of total DNA analysis, with an equivalent degree of discrimination.
Typing of Histoplasma capsulatum by restriction fragment length polymorphisms in a nuclear gene
Authors: Keath E J , Kobayashi G S , Medoff G
Published: January 2020

We previously described yps-3, a Histoplasma-specific nuclear gene probe useful in the identification of Histoplasma capsulatum. By using restriction fragment length polymorphisms (RFLPs) of DNA detected by the yps-3 gene and mitochondrial DNA, 76 clinical and soil isolates of H. capsulatum were classified. The majority of North American isolates obtained from endemic regions of the midwestern United States were members of the previously characterized class 2, although four clinical isolates from different patients with AIDS from that region were grouped in class 1 with the temperature-sensitive Downs strain. A Florida soil isolate (FLS1) was placed in class 4 on the basis of RFLP with both probes. Two American Type Culture Collection strains (G184B and G186B) from Panama were grouped into class 3 by this analysis. A group of five H. capsulatum isolates obtained from patients with AIDS in New York City were typed into a new class 5 on the basis of yps-3 polymorphisms; those organisms fell into two broad mitochondrial DNA patterns, designated 5b and 5c. Two new isolates from Panama were also members of this broad yps-3 class 5 group, but they exhibited a distinct mitochondrial DNA profile (class 5a). A sixth class was detected in DNA obtained from a patient with AIDS from Panama; that DNA had unique RFLP profiles with respect to both probes. These observations suggest that the Histoplasma-specific yps-3 gene probe is a sensitive tool for typing H. capsulatum in clinical specimens. Additionally, these studies provide molecular support for the hypothesis that AIDS-associated histoplasmosis in nonendemic areas is due to reactivation of a previously acquired infection.
Beta-glucuronidase activities of fecal isolates from healthy swine
Authors: Jackson L , Langlois B E , Dawson K A
Published: January 2020

Research has shown that various percentages of fecal Escherichia coli isolates obtained from healthy subjects may be beta-glucuronidase negative. The ability to detect beta-glucuronidase activity among fecal E. coli isolates from healthy subjects may be affected by assay conditions. A study was conducted in which agar and broth media containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) were used to examine beta-glucuronidase activities of fecal isolates from healthy swine. Rectal swabs were plated on MacConkey agar plus 100 mg of MUG per liter (MAC-MUG) and incubated at 35 degrees C for 24 h. Each of 986 isolates picked from MAC-MUG was inoculated into duplicate tubes of lauryl tryptose broth plus 50 mg of MUG per liter (LT-MUG). One set of tubes was incubated at 35 degrees C and the other set of tubes was incubated at 44.5 degrees C. Gas production and hydrolysis of MUG, indicated by fluorescence when observed with UV light with a wavelength of 360 nm, were determined after incubation for 24 and 48 h. A higher percentage (P less than 0.01) of isolates was MUG positive at 44.5 degrees C than at 35 degrees C after 24 h of incubation in LT-MUG. A higher percentage (P less than 0.01) of isolates was MUG positive after 48 h than after 24 h of incubation at both 35 and 44.5 degrees C. A lower percentage of isolates (P less than 0.05) was observed to be MUG positive on MAC-MUG agar compared with their MUG reactions in LT-MUG at 35 and 44.5 degrees C. Approximately 89% of the isolates identified were beta-glucuronidase-positive E. coli. The largest proportion of MUG-positive E. coli was detected with LT-MUG at 35 degrees C after 48 h of incubation.
Detection of group C rotavirus antigens and antibodies in animals and humans by enzyme-linked immunosorbent assays
Authors: Tsunemitsu H , Jiang B , Saif L J
Published: January 2020

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect group (gp) C rotavirus antigens and antibodies. Both assays were confirmed to be specific for gp C rotavirus by using serogroup A, B, and C rotaviruses; hyperimmune antisera to these serogroups of rotaviruses; and paired serum specimens from animals infected with gp C rotaviruses. The ELISA for antigen detection reacted not only with porcine gp C rotaviruses but also with human and bovine gp C rotaviruses. Following experimental challenge of gnotobiotic pigs with porcine gp C rotavirus, the virus was found by ELISA in all diarrheic feces. A high prevalence of antibodies to gp C rotaviruses was detected in sera from adult pigs (93 to 97%) and cattle (47 to 56%) in the United States and Japan. However, no antibody to gp C rotavirus was detected in the sera (n = 20) of adult horses in the United States. In human sera from Hokkaido, Japan, 3% of children and 13% of adults possessed antibody to gp C rotaviruses. These results suggest that the ELISA that we developed may be useful for surveying gp C rotavirus infections in animals and humans. On the basis of serology, gp C rotavirus infections are common in pigs and cattle in the United States and Japan, but they occur at lower levels in humans from the Hokkaido area of Japan.
Evaluation of the E test by using selected gram-positive bacteria
Authors: Ngui-Yen J H , Bryce E A , Porter C , Smith J A
Published: January 2020

The E test (AB Biodisk NA Inc.) was compared with standard reference methods using the National Committee for Clinical Laboratory Standards's recommendations for determining the MICs of four selected antibiotics against 208 clinical isolates of gram-positive bacteria. These bacteria included 32 strains of Streptococcus pneumoniae, 25 strains of Enterococcus faecium, 20 strains of oxacillin-sensitive Staphylococcus aureus (OSSA), 96 strains of oxacillin-resistant S. aureus (ORSA), and 35 strains of coagulase-negative staphylococci. Evaluation included MIC accuracy within 1 dilution, reproducibility testing, and cost analysis. There was 94% agreement between the E test and the reference method in testing S. pneumoniae and penicillin G. There was 92% agreement with ampicillin and 100% agreement with vancomycin in testing E. faecium isolates. Accuracy of the oxacillin E test with staphylococci was significantly improved by the use of salt-supplemented Mueller-Hinton agar, for an agreement of 100% with coagulase-negative staphylococci and oxacillin-sensitive S. aureus and that of 85% with oxacillin-resistant S. aureus, with no major discrepancies. The E test with American Type Culture Collection isolates and clinical strains gave excellent reproducibility and was less costly than microdilution panels when used to test fewer than three antibiotics. The E test is a simple, reliable, reproducible, and cost-effective method for MIC determination for gram-positive organisms.
Retrospective evaluation of two latex agglutination tests for detection of circulating antigens during invasive candidosis
Authors: Herent P , Stynen D , Hernando F , Fruit J , Poulain D
Published: January 2020

Two latex agglutination tests for the detection of Candida antigens, Pastorex Candida (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and Cand-Tec (Ramco Laboratories, Inc., Houston, Tex.), were applied to 79 serum samples from 19 patients who were retrospectively selected on the basis of mycological and clinical evidence of C. albicans infection and the availability of serial serum samples taken near the date of a positive culture. The specificity in 60 control individuals was 100% for Pastorex and 98.3% for Cand-Tec. The tests scored positive for 10 (52.6%) and 9 (47.4%) patients, respectively. Pastorex detected antigen in only 3 of 12 patients (25%) with positive antibody detection tests, but was positive for all 7 patients (100%) who produced no or a low antibody response, suggesting that the test performs better in the absence of antibodies. However, the sensitivity of Pastorex also increased with the number of samples available per patient, which was lower for high-antibody-responder patients (2.8 versus 5.7). If the patients who provided only one or two serum samples were eliminated, the sensitivity of Pastorex rose to 76.9%. For the Cand-Tec, the sensitivity was not related to the presence of antibodies, nor was it related to the number of samples per patient. The observed antigenemia was transient with both Pastorex and Cand-Tec. Only 12.5% of the positive reactions occurred on the same serum sample, confirming that the two tests react with different antigens. A positive antigen test preceded other diagnostic indications for 6 of 10 Pastorex-positive patients and 5 of 9 Cand-Tec-positive patients.
Evaluation of a urease-based confirmatory enzyme-linked immunosorbent assay for diagnosis of Neisseria gonorrhoeae
Authors: Carballo M , Dillon J R , Lussier M , Milthorp P , Winston S , Brodeur B
Published: January 2020

A new urease-based enzyme-linked immunosorbent assay utilizing novel monoclonal antibodies was evaluated for the culture confirmation of Neisseria gonorrhoeae, with 270 isolates of N. gonorrhoeae, 56 isolates of diverse Neisseria spp., and 29 Moraxella isolates. The test was highly specific (100.00%) and sensitive (97.83%). No cross-reactions were observed with any of the Neisseria or Moraxella isolates tested. Fifty percent (3 of 6) of the false-negative results were obtained with isolates of serovar IA-4, a serovar rarely encountered in North America.
Proposed interpretive criteria and quality control parameters for testing susceptibility of Neisseria gonorrhoeae to beta-lactam-clavulanate combinations
Authors: Fuchs P C , Barry A L , Baker C N , Murray P R , Washington J A
Published: January 2020

To support future clinical studies, in vitro susceptibility tests were examined to determine whether Neisseria gonorrhoeae could be tested reliably against two beta-lactam-clavulanate combinations. All isolates that were tested appeared to be susceptible to amoxicillin and ticarcillin in combination with clavulanic acid. In the absence of resistant isolates, only a breakpoint for a susceptible category could be defined for agar dilution tests with amoxicillin-clavulanic acid (MIC of less than or equal to 2.0/1.0 micrograms/ml is tentatively proposed). For disk diffusion tests, a corresponding breakpoint zone diameter of greater than or equal to 28 mm is suggested. The validity of the breakpoints for penicillinase-negative penicillin-resistant strains awaits clinical data. Proposed quality control limits for testing amoxicillin-clavulanic acid by agar dilution and disk diffusion methods are a MIC of 0.25/0.125 to 1.0/0.5 micrograms/ml and zones of 30 to 40 mm in diameter for N. gonorrhoeae ATCC 49226, a MIC of 0.125/0.06 to 0.5/0.25 micrograms/ml for Staphylococcus aureus ATCC 29213, and zones of 30 to 38 mm for S. aureus ATCC 25923. Ticarcillin-clavulanate is currently tested against other species by preparing doubling dilutions of ticarcillin with a constant 2 micrograms of clavulanate per ml. By that method, all gonococci were susceptible to low concentrations. However, the amount of clavulanic acid that is included (2 micrograms/ml) will, by itself, inhibit many strains of N. gonorrhoeae. Consequently, the role of ticarcillin in the combination cannot be determined, and such tests are not recommended.
Growth of Porphyromonas gingivalis, Treponema denticola, T. pectinovorum, T. socranskii, and T. vincentii in a chemically defined medium
Authors: Wyss C
Published: January 2020

A chemically defined medium, OMIZ (Oral Microbiology and Immunology, Zürich)-W1 was developed. Medium OMIZ-W1 supports the long-term proliferation of a wide range of oral anaerobes, including representative strains of four Treponema species and Porphyromonas gingivalis. High concentrations of ascorbic acid and ammonium ions proved to be important for the growth of these organisms. T. denticola CD-1 grew in the absence of polyamines and long-chain fatty acids, T. pectinovorum and T. socranskii required polyamines, whereas T. vincentii depended on both polyamines and lecithin for growth. Specific requirements for purines and/or pyrimidines were detected, and these requirements could be used to distinguish Haemophilus-Actinobacillus group organisms. Some strains of P. gingivalis grew without vitamin K, while others were not satisfied by menadione but required its precursor 1,4-dihydroxy-2-naphthoic acid. Protoporphyrin IX or hemin equally satisfied the porphyrin requirements of P. gingivalis and Bacteroides forsythus, whereas ferrous sulfate was more efficiently used as a source of iron than was hemin. The cellular cohesiveness of P. gingivalis increased with high concentrations of hemin in the growth medium. Prevotella intermedia, B. forsythus, and several strains of P. gingivalis were more fastidious and required a protein or serum supplement to grow in medium OMIZ-W1.
Confirmatory polymerase chain reaction testing for Chlamydia trachomatis in first-void urine from asymptomatic and symptomatic men
Authors: Mahony J B , Luinstra K E , Sellors J W , Jang D , Chernesky M A
Published: January 2020

First-void urine specimens from 683 men (592 without symptoms) were tested for Chlamydia trachomatis by a polymerase chain reaction (PCR) with KL1 and KL2 plasmid primers and by a Chlamydiazyme enzyme immunoassay (EIA). Thirty-seven specimens were confirmed to be positive by using the EIA blocking reagent and a second set of plasmid primers (T1 and T2). By comparing unconfirmed PCR results (KL1 and KL2 primers only) with the blocked Chlamydiazyme EIA results, the sensitivity and specificity of PCR were 100% (37 of 37 specimens) and 99.5% (643 of 646 specimens), respectively. Three additional specimens were negative by EIA but positive by PCR and were confirmed to be positive with primers T1 and T2. Two of the three specimens were from men with symptoms. The confirmatory PCR assay performed equally well in detecting positive specimens from symptomatic (31 of 31) and asymptomatic (9 of 9) men. Comparison of confirmatory testing of first-void urine specimens by PCR and EIA showed that PCR was 100% sensitive (40 of 40 specimens) and that the EIA was 92.5% sensitive (37 of 40 specimens) but that the assays were equally specific (100%).
Development and testing of a nonradioactive DNA oligonucleotide probe that is specific for Vibrio cholerae cholera toxin
Authors: Wright A C , Guo Y , Johnson J A , Nataro J P , Morris J G
Published: January 2020

An alkaline phosphatase-labeled oligonucleotide DNA probe (CTAP) that was specific for the cholera toxin gene (ctxA) was identified. All cholera toxin-producing strains of Vibrio cholerae, regardless of serotype, hybridized with the CTAP probe, while nontoxigenic strains from either environmental sources or from deletion or substitution mutations did not hybridize. Unlike the whole-gene probes for either ctxA or for the heat-labile toxin or Escherichia coli (eltA), this 23-base sequence did not hybridize with E. coli or with vibrios other than V. cholerae that produce related toxins. By using CTAP to identify colonies grown on nonselective medium, V. cholerae was enumerated at concentrations of 10(3) to 10(7)/g from stool samples of volunteers who had ingested V. cholerae O1 strain 569B. CTAP provides a specific and sensitive tool for diagnosis and environmental monitoring of cholera toxin-producing V. cholerae.
Blood spot screening and confirmatory tests for syphilis antibody
Authors: Stevens R , Pass K , Fuller S , Wiznia A , Noble L , Duva S , Neal M
Published: January 2020

We developed a blood spot test for syphilis antibody using enzyme-linked immunosorbent assay (ELISA) technology. Dried blood was eluted by buffered saline or, for a supplementary confirmatory test, by treponemal-antibody test diluent. Eluates were diluted in an absorption buffer (Calypte Biomedical, Berkeley, Calif.) and added to plate wells coated with cardiolipin antigen (ADI Diagnostics, Toronto, Ontario, Canada). The wells were washed and treated sequentially with an immunoglobulin G conjugate, buffer washes, and enzyme substrate. Substrate conversion was measured photometrically, and specimen reactivity was determined by reference to nonreactive controls. The optimum test protocol was established by tests of serum and plasma. The serum ELISA specificity with normal specimens was 98.9%. The sensitivity with sera from patients with undefined syphilis was 97.4%, that with sera from patients with documented primary and secondary disease was 100%, and that with sera from patients with early and late latent disease was 95.7%. The specificity of the spot test with donor blood was 94.2%, and its specificity with newborn blood was 94.9%. The sensitivity with 25 spots spiked with reactive sera was 96%. The seroprevalence rates for parturient women in one hospital were 6.01% according to spot tests of sera from 599 newborns and 6.81% according to Rapid Plasma Reagin tests of 499 maternal serum specimens. Seventy percent of infants born to 50 seropositive women were reactive by either the newborn spot or the Rapid Plasma Reagin serum test. The results show that blood spots may be used in seroprevalence or serodiagnostic studies, especially to identify women who are infected or to identify possible cases of congenital infection. The test provides for studies of children and adults when routine venipuncture and serum handling and storage are problematic.
Detection of human cytomegalovirus in plasma of AIDS patients during acute visceral disease by DNA amplification
Authors: Spector S A , Merrill R , Wolf D , Dankner W M
Published: January 2020

By using the polymerase chain reaction (PCR) amplification procedure, 19 (83%) of 23 plasma specimens obtained from individuals with AIDS and human cytomegalovirus (HCMV) visceral disease were found to be positive for plasma viremia as detected by PCR (PV-PCR), whereas 78% of cultures of peripheral blood leukocytes from the same samples were found to be positive. All 11 specimens prospectively obtained from individuals with acute HCMV disease were positive by PV-PCR. Plasma specimens from patients who received ganciclovir therapy rapidly became both culture and PV-PCR negative, and there was an excellent correlation between the two procedures. DNA detected by PV-PCR was unaffected by filtering plasma through a 0.2-microns-pore-size filter, although a conserved cellular gene, HLA-DQ alpha, was undetectable by PCR following filtration. HCMV DNA in plasma could be quantitated by PV-PCR by using endpoint serial dilutions, with detectable virus being present in 10(1) to 10(-2) microliters of plasma. A low titer of infectious virus could be detected in 2 of 11 plasma samples. The detection of HCMV DNA in plasma by PV-PCR promises to be a useful procedure for monitoring patients with AIDS suspected of having impending, acute, or recurrent HCMV visceral disease and suggests an additional route by which virus may disseminate in the immunocompromised host.