bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of epithelial interleukin-8 (IL-8) and neutrophil IL-8 receptor A in Escherichia coli-induced transuroepithelial neutrophil migration
Authors: Godaly G , Proudfoot A E , Offord R E , Svanborg C , Agace W W
Published: January 2020

Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8's ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.
Phase variation affects long-term survival of Bordetella bronchiseptica in professional phagocytes
Authors: Banemann A , Gross R
Published: January 2020

Several Bordetella bronchiseptica isolates were investigated for intracellular survival in macrophages. A significant number of viable bacteria of all strains could be recovered even after 96 h postinfection. In all cases bvg mutants of the B. bronchiseptica strains showed a significant survival advantage over the respective wild-type strains. The bacteria were already located in phagolysosomes early after uptake. Neither opsonization of the bacteria nor activation of the macrophages with gamma interferon or lipopolysaccharide prior to infection affected uptake and survival of the bacteria.
Adherence of Vibrio cholerae to cultured differentiated human intestinal cells: an in vitro colonization model
Authors: Bénitez J A , Spelbrink R G , Silva A , Phillips T E , Stanley C M , Boesman-Finkelstein M , Finkelstein R A
Published: January 2020

Choleragenic vibrios adhered to and multiplied on monolayers of the highly differentiated mucin-secreting cell line HT29-18N2. Their adherence followed first-order kinetics, was dependent on the concentration of vibrios, and was partially inhibited by lipopolysaccharide. Comparison of genetically modified vibrios showed that flagella, an active toxR gene, and the virulence cassette were not essential for initial binding. Inactivation of the hemagglutinin/protease increased binding. This highly differentiated human intestinal cell line provides a versatile new approach for studying major events occurring during intestinal colonization: adherence, multiplication, and detachment.
Analysis of expression of the alpha-toxin gene (hla) of Staphylococcus aureus by using a chromosomally encoded hla::lacZ gene fusion
Authors: Ohlsen K , Koller K P , Hacker J
Published: January 2020

The staphylococcal alpha-toxin (Hla) is a major virulence factor contributing to Staphylococcus aureus pathogenesis. To elucidate the conditions influencing hla expression, the determinant was fused to lacZ, the reporter gene coding for beta-galactosidase. The hla::lacZ fusion was integrated into the chromosome of the wild-type S. aureus strain Wood 46, leading to the variant Wood 46-3. Alpha-toxin expression was found to be dependent on temperature, showing a maximum at 42 degrees C. Furthermore, the indicator strain showed a growth phase-dependent hla regulation which was influenced by temperature. At 37 degrees C, induction of hla::lacZ expression occurred in the late exponential phase of growth, whereas at 42 degrees C, a strong induction was observed as early as the mid-exponential phase. These observations were verified by Northern blot analysis of hla mRNA and by Western blot (immunoblot) analysis of culture supernatants of strain Wood 46. It was additionally found that the induction of hla transcription at 42 degrees C was not coupled with higher concentrations of agr RNAIII, the effector molecule of the global regulator agr. Furthermore, expression of the alpha-toxin was repressed at a high osmolarity. It was also shown that oxygen is essential for hla expression and that cultivation of the S. aureus strain Wood 46-3 on solid medium and in the presence of carbon dioxide stimulated hla transcriptional activity.
Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells
Authors: Aragon V , Chao K , Dreyfus L A
Published: January 2020

Cytolethal distending toxin (CDT) is a newly described toxin produced by a number of enteropathogens, including Campylobacter jejuni, various Escherichia coli strains, and a few Shigella species. CDT induces distension and eventual death of a number of transformed cell lines. Here, we extend previous studies by demonstrating that morphological changes in CDT-treated Chinese hamster ovary cells are coincident with changes in cytoskeletal structure and an inhibition of cell proliferation. CDT-treated cells underwent a progressive accumulation of F-actin assemblies which microscopically resembled actin stress fibers. Accumulation of the stress fiber-like structures in CDT-treated cells was accompanied by an apparent blockage of cell division. Multinucleation was detected in some cells but did not constitute a significant feature of CDT action. Although toxin-treated cells failed to divide, cell viability remained high for the first 4 days following toxin treatment, as evidenced by trypan blue exclusion and neutral red uptake. [3H]thymidine incorporation studies on CDT-treated cells were consistent with a blockage of cell proliferation without a direct inhibition of DNA synthesis. Although the progression of toxin action developed slowly, a 2-min exposure to CDT resulted in an irreversible development of toxicity. Together, our data indicate that CDT affects F-actin assembly within target cells and may interrupt the regulation or function of cell cycle-dependent events leading to cytokinesis.
Increased susceptibility of RAG-2 SCID mice to dissemination of endodontic infections
Authors: Teles R , Wang C Y , Stashenko P
Published: January 2020

Specific immunity has been implicated in the pathogenesis of periapical lesions, although the extent to which these mechanisms are actually involved in either protection or destruction of the pulp-periapex complex is yet to be established. To investigate this question we compared periapical-lesion pathogenesis in RAG-2 severe combined immunodeficient (SCID) mice with immunocompetent control mice following surgical pulp exposure. In order to equalize the bacterial challenge, an infection protocol using Prevotella intermedia, Fusobacterium nucleatum, Peptostreptococcus micros, and Streptococcus intermedius was devised. The results demonstrated that after infection, the proportion of the root canal flora represented by the four pathogens was almost identical in both groups (39.9 and 42.2% for RAG-2 and immunocompetent control mice, respectively). The effects of abrogation of T- and B-cell mechanisms on periapical pathogenesis were then assessed. Approximately one-third of the RAG-2 mice developed endodontic abscesses, while no immunocompetent controls had abscesses, results which indicated regional dissemination of the infection. A similar incidence of abscesses was found in two additional experiments. Abscessed RAG-2 teeth had significantly larger periapical lesions than did nonabscessed RAG-2 teeth (P < or = 0.05) and exposed immunocompetent controls (P < or = 0.01), whereas nonabscessed RAG-2 teeth were not significantly different from those of exposed immunocompetent controls in periapical-lesion size. We conclude that B- and T-cell-mediated immunity protects the host from the dissemination of endodontic infections and that RAG-2 mice are more susceptible to infection-induced pulp-periapex destruction.
Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells
Authors: Gómez-Duarte O G , Dehio M , Guzmán C A , Chhatwal G S , Dehio C , Meyer T F
Published: January 2020

Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections.
Unwinding 20 Years of the Archaeal Minichromosome Maintenance Helicase
Authors: Kelman Lori M. , O’Dell William B. , Kelman Zvi
Published: January 2020

Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein.
Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology
Authors: Gunzer F , Karch H
Published: January 2020

We used the plasmid vector pGEX-2T for the expression of recombinant subunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl-beta-D-thiogalactopyranoside (IPTG) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.
Molecular analysis of multiple isolates of the major serotypes of group B streptococci
Authors: Fasola E , Livdahl C , Ferrieri P
Published: January 2020

Serotyping of clinical isolates is a widely used technique for epidemiologic study of group B streptococcal infections. However, serotyping cannot definitively determine epidemiologically related or unrelated isolates. We investigated the use of restriction endonuclease analysis (REA) with both conventional agarose gel electrophoresis (AGE) and pulsed-field gel electrophoresis (PFGE) in 50 isolates of the major serotypes of group B streptococci. Single digestion with HindIII and HaeIII and double digestion with HindIII and then EcoRI were used for conventional AGE, and digestion with SmaI was used for PFGE. The molecular profile of one strain was compared with those of the strains within the same serotype as well as with the profiles from strains of different serotypes. Among 10 type Ia, Ia/alpha, Ia/alpha+beta, and Ia/R1 isolates and depending on the restriction enzyme used, we found between five and six REA patterns by conventional AGE and seven by PFGE; among 4 type Ib/alpha+beta isolates we found 2 to 4 REA patterns by conventional AGE and 4 by PFGE; among 21 type II, II/alpha, II/beta, II/alpha+beta, and II/R4 isolates, we found 11 REA patterns by both AGE and PFGE; and among 14 type III, III/R1, and III/R4 isolates, we found from 7 to 12 different REA patterns by AGE and 10 by PFGE. In total, among 13 serotypes and one nontypeable strain, we found 29 to 31 REA patterns by conventional AGE and 33 by PFGE. A particular REA pattern within a serotype was different from the patterns found in the other serotypes, suggesting that REA analysis by using conventional AGE or PFGE is a sensitive method for analyzing genetic relatedness and diversity in group B streptococci and has potential value in molecular epidemiologic studies.
Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques
Authors: Brown D R , Bryan J T , Cramer H , Fife K H
Published: January 2020

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.
Prevalence of bacterial respiratory pathogens in the nasopharynx in breast-fed versus formula-fed infants
Authors: Kaleida P H , Nativio D G , Chao H P , Cowden S N
Published: January 2020

In several studies, breast-feeding has been associated with decreased frequency or duration of otitis media episodes. If a causal relationship exists, the mechanism of protection of breast-feeding has not been established. We hypothesized that infants who are breast-fed, compared with infants who are formula-fed, have a lower prevalence of nasopharyngeal colonization with the bacterial respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes) commonly isolated from the middle ear effusions of children with acute otitis media. In two private pediatric practices, we obtained specimens from the nasopharynx for culture from 211 infants at 1 month of age and from 173 of these infants at 2 months of age. A swab was left in place in the nasopharynx for 45 s and was then immediately transferred onto appropriate culture media. Exclusively breast-fed (n = 84) and exclusively formula-fed (n = 76) infants were similar regarding the number of persons in the household, the number of children in the household, the number of siblings in day care, and the proportion with a recent upper respiratory tract infection. The two groups did not differ significantly in the proportions found to have one or more respiratory pathogens at 1 month of age (10.7 versus 18.4%; P = 0.12) or 2 months of age (34.8 versus 35.1%; P = 0.57). We conclude that during the first 2 months after birth, the exclusive receipt of breast milk appears not to substantially influence the prevalence of nasopharyngeal colonization with common bacterial respiratory pathogens.
Comparison of quantitative cytomegalovirus antigenemia assay with culture methods and correlation with clinical disease
Authors: Landry M L , Ferguson D
Published: January 2020

Blood samples, obtained predominantly from human immunodeficiency virus-infected patients and solid-organ and bone marrow transplant recipients, were submitted to the clinical laboratory for detection of cytomegalovirus (CMV) and were processed by three methods: conventional culture, centrifugation culture, and CMV antigenemia assay with monoclonal antibodies (Clonab CMV; Biotest Diagnostic Corporation, Denville, N.J.) to CMV antigens. Of 496 blood samples tested, 107 were positive by one or more methods: 56 were positive by conventional culture, 27 were positive by centrifugation culture, and 97 were positive for CMV antigen (Ag) by the antigenemia assay. Forty-seven samples were positive by the CMV antigenemia assay only; in these samples, a mean of 12 Ag-positive cells was detected per 200,000 polymorphonuclear leukocytes examined. In contrast, samples positive by the CMV antigenemia assay and both culture methods had a mean of 193 Ag-positive cells, and samples positive by the CMV antigenemia assay and conventional culture alone had a mean of 157 Ag-positive cells. In the antigenemia assay, paraformaldehyde fixation resulted in superior cell morphology when compared with acetone fixation. Use of immunofluorescence staining reduced sample processing time and the complexity of reagent preparation in comparison with immunoperoxidase staining. Differences in the sensitivities between the immunofluorescence and immunoperoxidase staining techniques for detection of antigenemia were minor, with discrepant samples showing only one or two Ag-positive cells. Clinical disease was generally associated with high-level antigenemia, but exceptions were noted. The CMV antigenemia test is a rapid, quantitative assay that greatly facilitated the rapid diagnosis of CMV infection. However, quantitation of antigenemia is labor-intensive, requires processing of samples soon after collection, and does not always correlate with clinical disease in the individual patient.
Serologic evidence of canine and equine ehrlichiosis in northeastern United States
Authors: Magnarelli L A , Anderson J F
Published: January 2020

In a retrospective study, indirect fluorescent-antibody staining methods were used to detect immunoglobulins to Ehrlichia canis and Ehrlichia risticii in canine and equine sera that had originally been analyzed for antibodies to Borrelia burgdorferi. Analyses of 60 dog serum specimens collected in Connecticut and New York State during 1986 revealed antibodies to E. canis in 7 (11.7%) specimens; titration endpoints ranged from 1:40 to 1:320. Three of these dogs had anemia. Of the 187 equine serum specimens obtained in Connecticut during 1985 and analyzed by indirect fluorescent-antibody staining methods, 17 (9.1%) contained antibodies to E. risticii. Maximal antibody titers of 1:1,280 were recorded for serum specimens collected from three equids during May and July. We conclude that canine and equine ehrlichiosis coexist with Lyme borreliosis in Connecticut and the lower Hudson River Valley of New York State.
Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis
Authors: Belfaiza J , Postic D , Bellenger E , Baranton G , Girons I S
Published: January 2020

A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus Ixodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with MluI were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only fragments greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B. burgdorferi sensu lato.
Accelerated transmission of Lyme disease spirochetes by partially fed vector ticks
Authors: Shih C M , Spielman A
Published: January 2020

To determine how rapidly Lyme disease spirochetes (Borrelia burgdorferi) can be transmitted by partially fed vector ticks (Ixodes dammini), attached nymphs were removed from their hosts at various intervals post-attachment and subsequently permitted to re-feed to repletion on noninfected mice. We confirm previous reports that ticks deposit Lyme disease spirochetes in the skin of their hosts mainly after 2 days of attachment. Those that have been removed from a host within this interval can reattach and commence feeding. Spirochete-infected nymphs that have previously been attached to a host for 1 day become infectious to other hosts within another day. Noninfected nymphs acquire infection from spirochete-infected hosts within a day of attachment and become infectious to other hosts 3 to 5 days later. Virtually all ticks transmitted infection when reattaching after first feeding for 2 days. We conclude that partially fed nymphal ticks transmit spirochetal infection more rapidly than do ticks that have never been attached to a host and that infected ticks become infectious before they molt.
Characteristics of CDC group 1 and group 1-like coryneform bacteria isolated from clinical specimens
Authors: Funke G , Lucchini G M , Pfyffer G E , Marchiani M , von Graevenitz A
Published: January 2020

Fifteen strains of CDC group 1 coryneform and biochemically similar bacteria were isolated from clinical specimens. Of the 15 strains isolated, 11 were derived from abscesses and purulent lesions, mostly from the upper part of the body, and 3 were grown from blood cultures. Nine strains were associated with mixed anaerobic but no other aerobic flora. Seven strains exhibited the classical biochemical profile of CDC coryneform group 1; however, eight strains were unable to reduce nitrate and were called "group 1-like." Other reactions to differentiate CDC group 1 and group 1-like coryneform rods include alpha-hemolysis on human blood agar, fermentation of adonitol, and the presence of alkaline phosphatase. Fifteen strains showed marked CAMP reactions on different erythrocyte agars. Gas-liquid chromatography of volatile and nonvolatile fatty acids as well as cellular fatty acid patterns and the composition of cell wall components suggest that CDC group 1 and group 1-like coryneform bacteria do not belong to the genus Corynebacterium but possibly to the genus Actinomyces or Arcanobacterium. DNA-DNA hybridization studies revealed that group 1 and group 1-like strains represent different species.
Virological features of hepatitis C virus infection in hemodialysis patients
Authors: Silini E , Bono F , Cerino A , Piazza V , Solcia E , Mondelli M U
Published: January 2020

The clinical and epidemiological relevance of circulating antibodies to hepatitis C virus (HCV) in hemodialysis patients is uncertain, since clinical signs of infection are often mild or absent, with alanine aminotransferase (ALT) values that are virtually always normal, and liver biopsies are only rarely performed. Determination of HCV RNA in serum is therefore critical for distinguishing chronic HCV infection from previous exposure to the virus. We studied HCV viremia by reverse transcription polymerase chain reaction (RT-PCR) in the 5'-noncoding region of the viral genome in 77 dialysis patients who were screened for anti-HCV by a second-generation enzyme-linked immunosorbent assay (the enzyme immunoassay II; Ortho HCV, 2nd generation, Ortho Diagnostic Systems Raritan, N.J.) and a second-generation recombinant immunoblot assay (Chiron Corporation and Ortho Diagnostic Systems) and prospectively evaluated for ALT elevations over a period of 5 years. Of 77 patients tested, 29 (38%) had active infection as shown by a positive PCR assay result, and of these, 26 were anti-HCV positive. Although a good correlation was found between circulating anti-HCV and HCV RNA in serum, 10 (28%) of 36 anti-HCV-positive patients were HCV RNA negative by PCR, suggesting either low levels of viremia or past exposure to HCV and subsequent recovery. On the other hand, 3 (7.3%) of 41 anti-HCV-negative patients had HCV RNA in their sera, indicating seronegative HCV infection. The ALT level had no predictive value for HCV infection, because it was repeatedly normal in 18 (62%) of 29 viremic patients. HCV genotyping was also performed and indicated that all four known genotypes of HCV were present in our group. In conclusion, serological assays are reliable for detecting exposure to HCV in hemodialysis patients; however, direct identification of the viral genome is required to document current infection.
Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting
Authors: Corney B G , Colley J , Djordjevic S P , Whittington R , Graham G C
Published: January 2020

We compared random amplified polymorphic DNA (RAPD) fingerprinting with cross-absorption agglutination and restriction enzyme analysis for typing bovine leptospires. Using RAPD fingerprinting, we examined a number of Leptospira serovars, namely, hardjo genotypes bovis and prajitno, pomona, balcanica, tarassovi, swajizak, kremastos, australis, and zanoni, which are likely to be isolated from Australian cattle. Each serovar and genotype had a unique RAPD profile. Of 26 field isolates of Leptospira, 23 were identified as hardjo genotype bovis subtype A, 2 were identified as zanoni, and 1 was identified as pomona by RAPD fingerprinting, and their types were confirmed by cross-absorption agglutination and restriction enzyme analysis.
Detection of caprine arthritis-encephalitis virus by polymerase chain reaction
Authors: Reddy P G , Sapp W J , Heneine W
Published: January 2020

Detection of caprine arthritis-encephalitis virus (CAEV) infection in goats is currently limited to serologic testing or cell culture. We developed a polymerase chain reaction (PCR) assay to detect CAEV sequences in peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFC), and milk cells (MC) obtained from infected goats. Results were positive for 18 of 20 PBMC, 8 of 8 MC, and 5 of 5 SFC samples from seropositive goats, whereas 3 of 33 PBMC samples and none of 8 MC or 5 SFC samples from seronegative goats were positive. Two of the PCR-positive and seronegative goats seroconverted upon follow-up testing 2 months later. This PCR assay provides a useful method for detecting CAEV infection in goats.
Growth in serum-free medium improves isolation of Chlamydia pneumoniae
Authors: Maass M , Essig A , Marre R , Henkel W
Published: January 2020

Infectivity titers were determined for eight Chlamydia pneumoniae strains simultaneously grown in serum-free and serum-supplemented cell culture media. Use of serum-free medium resulted in a 10- to 50-fold increase in the susceptibility of HL cells to chlamydial infection. Comparative primary isolation of a wild-type strain also produced higher inclusion counts in a serum-free environment. Serum-free cultivation is recommended to increase the efficiency of C. pneumoniae isolation from clinical material and to permit elementary body purification without interference caused by serum components.
Reliability of two novel methods, Alamar and E test, for detection of methicillin-resistant Staphylococcus aureus
Authors: Novak S M , Hindler J , Bruckner D A
Published: January 2020

E test strips (AB Biodisk, Culver City, Calif.) tested on 2% NaCl-supplemented Mueller-Hinton agar and Alamar panels (Alamar, Sacramento, Calif.) correctly characterized 127 of 127 methicillin-resistant Staphylococcus aureus isolates and 100 of 100 methicillin-susceptible S. aureus isolates. These overnight antimicrobial susceptibility test systems reliably detect this clinically important resistance.
Serodiagnosis in early Lyme disease
Authors: Aguero-Rosenfeld M E , Nowakowski J , McKenna D F , Carbonaro C A , Wormser G P
Published: January 2020

Using a commercially available enzyme-linked immunosorbent assay (ELISA) and an immunoblot assay (IB), we tested sera from 100 patients with erythema migrans (EM) seen in 1991 a the Westchester County Medical Center Lyme Disease Diagnostic Center. Convalescent-phase sera were available from 59 patients. Fifty-five patients had EM of < 7 days' duration, 31 had EM of 7 to 14 days' duration, and 14 had EM of > 14 days' duration. During the acute phase of infection, 35 patients had a positive ELISA result and 43 had a positive IB result by the recently published criteria of Dressler et al. (F. Dressler, J. A. Whalen, B. N. Reinhardt, and A. C. Steere, J. Infect. Dis. 167:392-400, 1993) for interpretation of IB in patients with Lyme disease. A greater sensitivity of IB was observed in patients with EM of < 7 days' duration, as follows: 14 of 55 (25%) for IB versus 7 of 55 (13%) for ELISA (P = 0.144). Sera of all 14 patients with EM of > 14 days' duration were reactive by both tests, as follows: 13 positive and 1 equivocal by ELISA and 12 positive and 2 indeterminate by the IB. The band reactivity most frequently observed in the IB was to the 41- and 25-kDa antigens, the latter being the most frequent band observed in immunoglobulin M blots. Seroconversion was observed in 74 and 64% of evaluable patients by ELISA and IB, respectively, despite the use of antibiotic therapy.
Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test
Authors: Abe C , Hirano K , Wada M , Kazumi Y , Takahashi M , Fukasawa Y , Yoshimura T , Miyagi C , Goto S
Published: January 2020

The polymerase chain reaction (PCR) using oligonucleotides based on the repetitive sequence (IS986) of Mycobacterium tuberculosis as a primer and the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which combines an M. tuberculosis rRNA amplification method with the hybridization protection assay format, were evaluated for detection of M. tuberculosis in clinical samples. The detection limits of these two assay systems based on nucleic acid amplification for cultured M. tuberculosis were less than 10 cells per reaction. A total of 135 sputum specimens were examined by the two assay systems. The PCR and the MTD systems for detection of M. tuberculosis gave overall positivity rates of 84.2% (32 of 38) and 91.9% (34 of 37), respectively, as compared with 71.9% (23 of 32) by smear and 96.9% (31 of 32) by culture in the liquid medium MB-Check. Procedures for sample preparation used in the two methods were different. Although the sensitivities of the PCR and MTD appeared to be similar to that of culture with the MB-Check system, the two methods based on nucleic acid amplification should be very useful for rapid detection of M. tuberculosis infections without the long time required for culture of M. tuberculosis.
Reliability of the E test for detection of ampicillin, vancomycin, and high-level aminoglycoside resistance in Enterococcus spp
Authors: Schulz J E , Sahm D F
Published: January 2020

By comparison with agar dilution results, the E test was investigated for the ability to detect high-level aminoglycoside (gentamicin and streptomycin), ampicillin, and vancomycin resistance among strains representing six enterococcal species. For ampicillin and vancomycin, disk diffusion results also were obtained. No false high-level aminoglycoside resistance occurred, and no false gentamicin susceptibility was noted. With the high-range streptomycin E test (2,048 micrograms), 24% of the 38 resistant strains were falsely susceptible. However, these discordances could likely be reconciled by adjustments in incubation duration and by using broth microdilution rather than agar screen breakpoint criteria, or by using the lower-range (1,024-micrograms) strip. For ampicillin, category results obtained by E test and disk diffusion showed good agreement with agar dilution; E test MICs were generally comparable to agar dilution MICs. The E test was more sensitive than disk diffusion for detecting vancomycin-intermediate strains, but for these strains and those exhibiting low-level vancomycin resistance (MIC, 32 to 128 micrograms/ml), disk diffusion and E test inhibition zones must be interpreted with caution. Given the reliability of E test for detecting resistance to anti-enterococcal agents, the decision to use this method should be based on convenience, cost, testing frequency, and satisfaction with currently used methods.
Reproducibility of lysis-centrifugation cultures for quantification of Mycobacterium avium complex bacteremia
Authors: Havlir D , Kemper C A , Deresinski S C
Published: January 2020

While quantitative mycobacterial blood cultures have been accepted as the standard for evaluating response to various Mycobacterium avium complex (MAC) treatment regimens, variability in this methodology has not been evaluated in a rigorous fashion. We thus studied the reproducibility of quantitative MAC cultures by a lysis-centrifugation culture system within and among five institutions. To measure the intralaboratory variation in mycobacterial colony counts, colony counts from duplicate blood specimens collected from 52 AIDS patients with MAC bacteremia were determined. Colony counts ranged from 0 to 50,000 CFU/ml. Nonparametric analyses revealed there was no significant difference in colony counts between the 52 duplicate specimens. The agreement between the intralaboratory paired specimens, as measured by the intraclass correlation coefficient, was 0.997. To measure the interlaboratory variation, multiple 10-ml aliquots from 12 patients were distributed to five institutions and processed within 24 to 32 h by lysis-centrifugation. For the 12 specimens distributed to the five laboratories, two-way analysis of variance for repeated measures revealed no significant difference in an individual patient's colony counts between laboratories (P > 0.2). We conclude that quantitation of mycobacterial colony counts by the lysis-centrifugation system is reproducible within and between institutions. Clinical trials evaluating response to therapeutic interventions for MAC can use multiple laboratories for quantitation of mycobacteremia. Furthermore, a 24- to 32-h delay in processing appeared to have no impact on reproducibility.
Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis
Authors: van der Zee A , Agterberg C , Peeters M , Schellekens J , Mooi F R
Published: January 2020

A polymerase chain reaction (PCR) assay which allows the simultaneous detection and discrimination of the two causative agents of pertussis, Bordetella pertussis and Bordetella parapertussis, was developed. Primer pairs were based on insertion sequence elements IS481 and IS1001. IS481 is specific for B. pertussis and is present in about 80 copies per cell, while IS1001 is specific for B. parapertussis and is found in 20 copies per cell. An internal control was included in the PCR assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. Discrimination of amplified DNA derived from the internal control, B. pertussis, or B. parapertussis was accomplished by differential spacing of the primers. The sensitivity of the combined PCR method was found to be very high and allowed the detection of one cell of either pathogen. The usefulness of the method was investigated by using a limited number of clinical samples derived from patients with serologically proven pertussis.
Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis
Authors: Da Silva A J , Piuvezam M R , de Moura H , Maddison S , Peralta J M
Published: January 2020

A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract.
Use of induced sputum specimens for microbiologic diagnosis of infections due to organisms other than Pneumocystis carinii
Authors: Fishman J A , Roth R S , Zanzot E , Enos E J , Ferraro M J
Published: January 2020

The optimal diagnostic approach to pneumonia provides a rapid microbiologic identification of pulmonary pathogens by the least invasive means. The technique of sputum induction has been useful in the evaluation of patients with Pneumocystis carinii or mycobacterial pneumonia. It is not known whether induced sputum samples are preferable for the detection of pathogens other than P. carinii or mycobacteria. Microbiologic yields were evaluated from identically processed induced and conventional sputum samples collected from 509 consecutive patients. No statistically significant differences were found between the microbiologic yields of induced and spontaneous sputum samples. Bacterial pathogens were isolated in 19.6% of induced and 23.5% of routine specimens. Mycobacteria were cultured from 13.1% of routine and 9.4% of induced specimens. Non-Candida albicans fungi grew from 24% of routine and 20% of induced specimens. The process of sputum induction with aerosolized hypertonic saline did not alter either the purulence or the bacterial quantitation of Gram-stained sputum specimens. Sputum induction has been useful for the cytologic diagnosis of malignancy, for the diagnosis of pneumocystosis and tuberculosis, and in patients unable to spontaneously produce sputum samples. The use of induced sputum samples for the diagnosis of other infections may not be necessary when routine sputum specimens are available.
Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units
Authors: Leonard R B , Nowowiejski D J , Warren J J , Finn D J , Coyle M B
Published: January 2020

During a 14-month period, a unique strain of Corynebacterium striatum that produces a diffusible brown pigment was isolated from purulent sputa of nine patients and from nonrespiratory sites of two additional patients. Seven nonpigmented clinical isolates from the same period and three reference strains of C. striatum were compared with the brown isolates. Most patients had multiple sputum cultures with no coryneforms before the brown strain emerged, suggesting that the organism was hospital acquired. DNA restriction fragment patterns and Southern hybridization with the att site probe of Corynebacterium diphtheriae indicated that the brown isolates were a single strain which was distinct from the heterogeneous nonpigmented strains. A common source for the brown C. striatum was not recognized, although all of these patients were located in two adjoining intensive care units. All of the brown isolates, three of the nonpigmented clinical isolates, and two reference strains had positive CAMP reactions with Staphylococcus aureus, which has not been reported for C. striatum prior to this study.
Diagnostic value of anti-Candida enolase antibodies
Authors: van Deventer A J , van Vliet H J , Hop W C , Goessens W H
Published: January 2020

An immunodominant antigen with enolase enzyme activity was purified and used for the development of an assay to detect antibodies directed against this antigen in sera from patients with either invasive candidiasis or Candida colonization. The Au enzyme-linked immunosorbent assay established with the Candida enolase antigen was able to discriminate significantly between invasive candidiasis and colonization in both immunocompetent and immunodeficient groups of patients. The test had a sensitivity of 50% and a specificity of 86% in the immunocompetent patient group. In the immunodeficient patient group, a sensitivity of 53% and a specificity of 78% were established. Antibody levels determined by a counterimmunoelectrophoresis assay with the same set of sera resulted in a better sensitivity for sera from the immunocompetent patient group but a lower specificity, i.e., 80 and 29%, respectively. The counterimmunoelectrophoresis assay of sera from the immunodeficient patient group was not able to discriminate significantly between invasive candidiasis and colonization. With the use of more serum from each patient, the sensitivity of the antibody detection assays increased, while the specificity was maintained. The increase, however, was not statistically significant. Combining the results of the antibody assays with antigen titers obtained by the Cand-Tec assay did not improve the predictive value with respect to invasive candidiasis, as determined by multivariance regression analysis. Furthermore, it was demonstrated by performance of Western blots (immunoblots) that sera from patients as well as a rabbit antiserum cross-reacted with the Candida enolase and baker's yeast enolase enzyme. However, by tandem crossed immunoelectrophoresis it was demonstrated that the antibodies were directed toward different epitopes of the antigen.
Utility of quantitative enzyme immunoassay reactivity for predicting human immunodeficiency virus seropositivity in low- and high-prevalence populations
Authors: Hou X , Breese P L , Douglas J M
Published: January 2020

To assess the utility of quantitative enzyme immunoassay (EIA) reactivity for predicting human immunodeficiency virus seropositivity, we evaluated 22,823 serum samples from homo- and bisexual men, heterosexual intravenous drug users, and other heterosexuals with initial screening by EIA, retesting of reactive samples in duplicate, and confirmatory Western blot (immunoblot) testing. Quantitative EIA reactivity was determined by a mean of the optical density ratio of the three assays performed for each reactive specimen. A total of 1,773 samples (7.8%) were repeatedly reactive, and 1,747 (7.7%) were confirmed Western blot positive. All 26 EIA-reactive-Western blot-negative samples had low-level EIA reactivity (ratio < 2.2), while most (86%) of the Western blot-positive samples had high-level reactivity (ratio, > 3.0). The positive predictive value for samples with moderate-to-high-level EIA reactivity (ratio, > 2.2) was 100% for all risk groups. These results support the value of quantitative EIA reactivity in predicting human immunodeficiency virus seropositivity and suggest that confirmatory testing of specimens with high-level reactivity is not necessary in all situations.
Prevalence of hepatitis C virus genotypes in Italy
Authors: Pistello M , Maggi F , Vatteroni L , Cecconi N , Panicucci F , Bresci G P , Gambardella L , Taddei M , Bionda A , Tuoni M
Published: January 2020

Hepatitis C viruses (HCV) present in 110 Italian patients were characterized by genotype-specific PCRs. Among the 65 cases of community-acquired hepatitis, HCV genotype II was dominant (60%), followed by genotypes IV (15%), III (11%), and I (3%). Among the 45 hemophilia-associated cases, the distribution of the four HCV genotypes was markedly different: genotype I was the most prevalent (61%), followed by genotypes II (25%), III (4%), and IV (2%). Double infections were observed in eight patients. Two HCV remained unclassified. For the 45 community-acquired cases from which a liver biopsy was available, genotype II was associated with more severe liver damage than the other types.
Morphological diversity of Blastocystis hominis in sodium acetate-acetic acid-formalin-preserved stool samples stained with iron hematoxylin
Authors: MacPherson D W , MacQueen W M
Published: January 2020

The objective of this investigation was to study the morphological characteristics of Blastocystis hominis in sodium acetate-acetic acid-Formalin-preserved stool samples. Routinely processed samples were examined for morphological detail, including size, shape, nuclear detail, and central body characteristics. Morphological findings revealing the importance of recognizing B. hominis in the diagnostic laboratory are described.