bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of the humoral immune response against Plasmodium falciparum rhoptry-associated proteins 1 and 2
Authors: Stowers A , Taylor D , Prescott N , Cheng Q , Cooper J , Saul A
Published: January 2020

Naturally occurring antibody responses to Plasmodium falciparum rhoptry-associated proteins 1 and 2 (RAP-1 and RAP-2) were measured with recombinant and parasite-derived forms of the antigens. For comparative purposes, responses to multiple forms of three other malarial antigens were also examined. The sera of 100 Papua New Guineans were screened for antibodies. Eighty-six and 82% of individuals over 30 years of age had antibodies that recognized parasite-derived RAP-1 and RAP-2, respectively. Importantly, we found that recombinant and native antigens share linear epitopes seen by the human immune system; thus, the recombinant proteins may be adequate human immunogens. However, antibodies affinity purified on recombinant RAP-1 reacted with other antigens in addition to parasite-derived RAP-1. Thus, the antigenicity of RAP-1 may have been overestimated previously. The recognition of RAP-1 and RAP-2 correlated with age and with the recognition of recombinant forms of the ring-infected erythrocyte surface antigen, merozoite surface protein 1, and merozoite surface antigen 2 (MSA2) antigens. Antibodies to these antigens appear to be generated in response to the total exposure to malaria of the host. Antibodies to conserved regions of MSA2 had stronger correlations with both age and the recognition of other antigens than did the full-length recombinant MSA2 molecule. In contrast to results with the other antigens, there was no significant difference in the ages of individuals with a certain antibody titer to the full-length recombinant or parasite-derived MSA2 molecule, but antibodies to these two antigens did correlate with parasitemia. For all antigens tested, antibody levels after two infections can approach the peak levels of antibodies obtained in immune individuals.
Pathologic changes during acute Q fever: influence of the route of infection and inoculum size in infected guinea pigs
Authors: La Scola B , Lepidi H , Raoult D
Published: January 2020

As assessed by both standard histological staining and immunochemistry, intraperitoneal inoculation of C. burnetii in guinea pigs led to pathologic changes mainly in the liver, whereas intranasal inoculation led to pathologic changes mainly in the lungs. Myocarditis and positive blood cultures were observed only in those animals which received the highest inoculum. We therefore conclude that both the route of infection and the size of the inoculum influence clinical expression in acute Q fever.
Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation
Authors: Sporn L A , Sahni S K , Lerner N B , Marder V J , Silverman D J , Turpin L C , Schwab A L
Published: January 2020

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is an obligate intracellular bacterial organism that infects primarily the vascular endothelial cells (EC). A component of the EC response to infection is transcriptional activation, which may contribute to the thrombotic and inflammatory consequences of disease. In this study, we explore R. rickettsii-induced activation of the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors involved in early transcriptional responses to injurious stimuli. Two NF-kappaB species were activated by infection and reacted with a double-stranded oligonucleotide probe corresponding to the kappaB binding domain of the murine kappa light-chain gene enhancer. Gel supershift analysis demonstrated the reactivity of these complexes with antibodies against p65 and p50, and the induced species were tentatively identified as p50-p50 homodimers and p50-p65 heterodimers. Semiquantitative reverse transcription-PCR analysis revealed dramatic increases in the steady-state levels of mRNA coding for the inhibitory subunit of NF-kappaB (IkappaB alpha), transcription of which is enhanced by the binding of NF-kappaB within the IkappaB alpha promoter region. NF-kappaB activation was first detected 1.5 h following infection and was biphasic, with an early peak of activation at approximately 3 h, a return to baseline levels at 14 h, and even higher levels of activation at 24 h. It is likely that NF-kappaB activation requires cellular uptake of R. rickettsii, since treatment of EC with cytochalasin B during infection to block entry inhibited activation by only 70% at 3 h. R. rickettsii-induced activation of NF-kappaB may be an important controlling factor in the transcriptional responses of EC to infection with this obligate intracellular organism.
Identification and characterization of S fimbria-binding sialoglycoproteins on brain microvascular endothelial cells
Authors: Prasadarao N V , Wass C A , Kim K S
Published: January 2020

We have previously shown that S-fimbriated Escherichia coli binds brain microvascular endothelial cells (BMEC) via a lectin-like activity of SfaS adhesin specific for NeuAc alpha2,3-galactose; however, BMEC molecules bearing these epitopes have not been identified. In the present study, we showed that the expression of S fimbriae conferred a three-fold increase in adhesion of E. coli to cow, human, and rat BMEC but did not enhance E. coli adhesion to systemic vascular endothelial cells such as human umbilical vein endothelial cells and human aortic arterial endothelial cells. Two BMEC-binding molecules for S fimbriae were identified as 65 (major)- and 130 (minor)-kDa sialoglycoproteins by S fimbria immunoblotting and were purified from bovine BMEC by wheat germ agglutinin and Maackia amurensis lectin (specific to NeuAc alpha2,3-galactose) affinity chromatography. The 65-kDa BMEC glycoprotein showed effective inhibition of S fimbria-mediated binding of E. coli to BMEC. Polyclonal antibodies raised against the mixture of 65- and 130-kDa proteins reacted to 65-kDa protein present only on BMEC, not on systemic vascular endothelial cells. Immunoprecipitation of biotinylated BMEC membrane proteins and immunocytochemistry studies of BMEC with anti-S fimbria-binding protein antibodies revealed that the 65-kDa protein is a surface protein. The N-terminal amino acid sequence of 65- and 130-kDa proteins showed no significant sequence homology with any other known proteins. These findings suggest that 65- and 130-kDa proteins represent novel sialoglycoproteins involved in the binding of S-fimbriated E. coli to BMEC.
Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230
Authors: Healer J , McGuinness D , Hopcroft P , Haley S , Carter R , Riley E
Published: January 2020

Antibodies to the sexual-stage surface antigens of Plasmodium falciparum, Pfs230 and Pfs48/45, can abolish the infectivity of gametes to mosquitoes; these antigens have been proposed as candidates for inclusion in a malaria transmission-blocking vaccine. One possible mechanism of antibody-mediated transmission blocking is complement-mediated gamete lysis. We have used a panel of human sera from geographically distinct regions where malaria is endemic to investigate whether this may be a mechanism of naturally acquired transmission-blocking immunity to P. falciparum. By immunoprecipitation, we have shown that antibody recognition of Pfs230 and Pfs48/45 is limited, despite universal exposure to P. falciparum gametocytes. In vitro complement-mediated lysis of P. falciparum gametes was positively associated with the presence of antibodies to Pfs230 but not with antibodies to the N-terminal region of the precursor molecule (Pfs260), which is shed from the gametocyte surface at the time of gametogenesis. Similarly, antibodies to two other gametocyte-specific proteins, Pfs48/45 and Pfg27/25, were not associated with gamete lysis. All sera which mediate gamete lysis contain immunoglobulin G1 (IgG1) and/or IgG3 antibodies to gamete surface proteins as determined by an enzyme-linked immunosorbent assay. These data suggest that Pfs230 is a major target of complement-fixing antibodies which may be important for antibody-mediated transmission-blocking immunity.
Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes
Authors: Roberts F A , Richardson G J , Michalek S M
Published: January 2020

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.
Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature
Authors: Abe A , Kenny B , Stein M , Finlay B B
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions. Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis. At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions. However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized. To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127. The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity). However, RDEC-1 EspB was identical to that of enterohemorrhagic E. coli serotype O26. Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells. Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion. Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits.
Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide
Authors: Jungi T W , Sager H , Adler H , Brcic M , Pfister H
Published: January 2020

The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.
Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in human alveolar macrophages
Authors: Nozaki Y , Hasegawa Y , Ichiyama S , Nakashima I , Shimokata K
Published: January 2020

We demonstrated that products of the L-arginine-dependent pathway of human alveolar macrophages (AM) effectively kill the Mycobacterium bovis bacillus Calmette-Guérin (BCG) in vitro. The formation of products was triggered by inoculation with BCG itself. Many reports have shown that activated rodent AM could produce an amount of nitric oxide (NO) sufficient to suppress the growth of mycobacteria. However, there have been no definitive results as to whether human AM might have the NO-dependent mechanism for the killing of mycobacteria. Therefore, we have undertaken some experiments to answer this question. Immunofluorescence assays showed an increased production of inducible nitric oxide synthase (iNOS) and peroxynitrite in BCG-inoculated AM from patients with pulmonary fibrosis. Reverse transcriptase-PCR also revealed the higher expression of iNOS-coding mRNA. Colony assays demonstrated that these human AM effectively killed BCG in their cytoplasm. However, treatment of AM with N(G)-monomethyl-L-arginine monoacetate resulted in markedly reduced killing activity. These results clearly show that BCG-induced NO and its reactive product with the oxygen radical peroxynitrite could play an important role in the killing of BCG in human AM.
The Oms66 (p66) protein is a Borrelia burgdorferi porin
Authors: Skare J T , Mirzabekov T A , Shang E S , Blanco D R , Erdjument-Bromage H , Bunikis J , Bergström S , Tempst P , Kagan B L , Miller J N , Lovett M A
Published: January 2020

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.
Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression
Authors: Schuch R , Maurelli A T
Published: January 2020

Expression of the predominantly plasmid-encoded virulence regulon of Shigella flexneri 2a is induced by growth at 37 degrees C and repressed by growth at 30 degrees C. During growth at 37 degrees C, spontaneous S. flexneri mutants arise which have undergone virulence plasmid curing or rearrangement and no longer display the virulent phenotype. In the laboratory, the unstable nature of the virulence plasmid causes complete loss of virulence in a growing population. We have undertaken an analysis of virulence plasmid instability, classifying events which produced individual avirulent derivatives within a virulent population and identifying the factor(s) which controlled conversion. Multiplex PCR analysis of DNA obtained from spontaneous avirulent derivatives indicated that virF and virB were deleted or otherwise inactivated in over 97% of the isolates. The virF and virB loci encode regulatory proteins required for transcriptional activation of the virulence regulon. Inactivation of these key regulatory loci in the vast majority of avirulent derivatives which arose during growth at 37 degrees C suggested that virulence gene expression induced virulence plasmid instability. Consistent with this hypothesis, we observed stable virulence plasmid maintenance during growth of a wild-type strain at 30 degrees C where virulence gene expression was repressed. The virulence plasmid was also stably maintained in virF and virB mutants grown at 37 degrees C. Conversely, virulence plasmid destabilization was induced at 30 degrees C and accelerated at 37 degrees C through expression of VirF or VirB from multicopy plasmids. These results indicate that exposure of S. flexneri to conditions favoring induction of the virulent phenotype also favor its loss. The significance of this paradox of Shigella pathogenicity is discussed.
Role of interleukin 12 in experimental neonatal sepsis caused by group B streptococci
Authors: Mancuso G , Cusumano V , Genovese F , Gambuzza M , Beninati C , Teti G
Published: January 2020

Cytokines are suspected to play an important role in systemic infections by group B streptococci (GBS), an important cause of neonatal sepsis. This work was undertaken to determine if interleukin 12 (IL-12) is produced in mouse pups infected with GBS and has a role in this sepsis model. IL-12 elevations were measured by both an enzyme-linked immunosorbent assay and a bioassay in plasma samples obtained from 12 to 72 h after GBS challenge. Pretreatment with neutralizing anti-IL-12 antibodies significantly increased lethality and blood CFU (P < 0.05). Conversely, either prophylactically or therapeutically administered recombinant IL-12 (rIL-12) significantly improved survival time and decreased blood CFU. Since these beneficial effects were associated with increased spleen gamma interferon (IFN-gamma) production, we examined whether the latter cytokine mediated the observed rIL-12 effects. Pretreatment with neutralizing anti-IFN-gamma monoclonal antibodies significantly counteracted the beneficial effects of rIL-12 on lethality. Our data indicate that rIL-12 is a possible candidate for treatment of GBS sepsis and that its activities in this model are at least partially mediated by IFN-gamma.
Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits
Authors: Von Moll L K , Cantey J R
Published: January 2020

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.
Allelic variation in Streptococcus pneumoniae autolysin (N-acetyl muramoyl-L-alanine amidase)
Authors: Gillespie S H , McHugh T D , Ayres H , Dickens A , Efstratiou A , Whiting G C
Published: January 2020

The lytA gene encoding the autolysin of Streptococcus pneumoniae may be a virulence determinant. Single-strand conformational polymorphism analysis demonstrated heterogenicity throughout the gene in clinical isolates and strains from the clonal serotypes 7 and 14. Sequence analysis of part of the choline-binding domain showed that in two isolates four amino acid substitutions occurred.
A nonsubstituted primary hydroxyl group in position 6' of free lipid A is required for binding of lipid A monoclonal antibodies
Authors: Brade L , Engel R , Christ W J , Rietschel E T
Published: January 2020

Lipid A monoclonal antibodies, which require for binding the presence of the bisphosphorylated D-glucosamine disaccharide lipid A backbone, were tested against synthetic lipid A precursor Ia and compound B 1047 by enzyme immunoassay. The last-named compound is a precursor Ia analog with a methoxy instead of a hydroxy group at C6' and was chosen to determine why these antibodies failed to recognize the bound lipid A present in lipopolysaccharide (LPS). Whereas all antibodies tested bound to precursor Ia, none of them bound to compound B 1047 or Escherichia coli Re LPS to a significant extent. Compared to the natural substituent at C6', i.e., 3-deoxy-D-manno-octulosonic acid (Kdo), the methoxy group is neither bulky nor charged. Thus, the data suggest that it is not hindrance by Kdo but rather the generation of a neoantigen that endows lipid A with immunoreactivity upon liberation from LPS by acid hydrolysis.
Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods
Authors: Huang M B , Gay T E , Baker C N , Banerjee S N , Tenover F C
Published: January 2020

The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of < or = 2 micrograms/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of > or = 4 micrograms/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence of added salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were > 17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.9%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of > or = 18.3%. We recommended the addition of 2% NaC1 to Mueller-Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaC1 should not be added for the disk diffusion test.
Use of monoclonal antibodies to distinguish pathogenic Naegleria fowleri (cysts, trophozoites, or flagellate forms) from other Naegleria species
Authors: Sparagano O , Drouet E , Brebant R , Manet E , Denoyel G A , Pernin P
Published: January 2020

Monoclonal antibodies (MAbs) reactive to the pathogenic amoeba Naegleria fowleri were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, Western blotting (immunoblotting), and radioimmunoprecipitation assay (RIPA). Two MAbs (3A4 and 5D12) showed reactivity by ELISA with all N. fowleri strains tested and no reactivity with the five other Naegleria species, N. lovaniensis, N. gruberi, N. australiensis, N. jadini, and N. andersoni. These MAbs reacted with the three morphological forms of N. fowleri (trophozoites, cysts, and flagellates). The reactivity on Western blots was suppressed by treatment with metaperiodate, suggesting a carbohydrate epitope. Differences in reactivity patterns between trophozoites and cysts observed with radioimmunoprecipitation assay might reflect differences in biological properties. The formalin stability of the epitope may be useful in detecting N. fowleri in fixed biopsies and in investigating the pathological process.
Simple and rapid two-step polymerase chain reaction for diagnosis of Pneumocystis carinii infection
Authors: Tamburrini E , Mencarini P , De Luca A , Antinori A , Visconti E , Ammassari A , Ortona L , Ortona E , Siracusano A , Vicari G
Published: January 2020

Two amplification steps were made to detect Pneumocystis carinii DNA by polymerase chain reaction (PCR). pAZ102-E and pAZ102-H (standard PCR), pAZ102-L2 (sense), and pAZ102-E (antisense) (two-step PCR) were used as primers. The amplification products were analyzed by ethidium bromide. After the two-step PCR, ethidium bromide detected all samples positive by oligohybridization after one amplification step. Our two-step PCR is a rapid, cost-effective, and clinically suitable method for the detection of P. carinii infection.
Use of the Quantitative Buffy Coat system for detection of parasitemia in patients with babesiosis
Authors: Mattia A R , Waldron M A , Sierra L S
Published: January 2020

Quantitative Buffy Coat analysis and blood smears were performed on a total of 47 blood samples. The technique showed 100% correlation with the blood smears in 9 samples containing babesia and 10 samples containing malaria, with some differential features distinguishing the two infections. Quantitative Buffy Coat analysis provides a simple and rapid method for the detection of parasitemia in cases of babesiosis.
Detection of antibody to recombinant Norwalk virus antigen in specimens from outbreaks of gastroenteritis
Authors: Monroe S S , Stine S E , Jiang X , Estes M K , Glass R I
Published: January 2020

Norwalk virus and other small round-structured viruses are commonly associated with outbreaks of gastroenteritis. We used a recently described recombinant-expressed Norwalk virus (rNV) capsid protein in enzyme immunoassays to quantitatively measure immunoglobulin G (IgG) and IgA to Norwalk virus in serum pairs from patients involved in outbreaks of gastroenteritis. The outbreaks previously were classified, on the basis of the results of a blocking antibody assay, as Norwalk virus negative, serologically intermediate, or Norwalk virus positive. The rNV IgG assay was more sensitive than the blocking assay for detecting IgG to Norwalk virus in serum from patients in all outbreak classes. There was 79% concordance between seroconversions detected by the blocking antibody assay and those detected by the rNV IgG assay. The rNV IgA assay detected seroconversions to Norwalk virus primarily in patients involved in outbreaks previously classified as Norwalk virus positive.
Life cycle of the human and animal oomycete pathogen Pythium insidiosum
Authors: Mendoza L , Hernandez F , Ajello L
Published: January 2020

Pythium insidiosum, the etiologic agent of pythiosis insidiosii, causes life-threatening infections in humans and animals. Previous studies of the epidemiology of this disease hypothesized about the possible life cycle of this oomycete. Details, however, were not provided on the steps required to cause infection. We investigated the life cycle of P. insidiosum by inoculating pieces of equine skin and plant leaves and then studying the ensuing events with a scanning electron microscope. Our observations revealed that zoospores had a strong tropism for skin tissue, horse and human hair, and water lily and grass leaves and a weak attraction to a variety of other leaves. Encysted zoospores were observed on the favored leaves and skin. There they produced germ tubes and later abundant hyphal filaments that penetrated leaf tissues. Young sporangia had compact, thick walls. The sporangial wall was reduced to a fragile membrane when the sporangia had produced well-differentiated biflagellate zoospores. The encysted zoospores secreted an amorphous material that permitted the zoospores to adhere to skin and plant tissues. On the basis of these findings, a model to explain the life cycle of P. insidiosum is proposed.
Bactericidal, virucidal, and mycobactericidal activities of reused alkaline glutaraldehyde in an endoscopy unit
Authors: Mbithi J N , Springthorpe V S , Sattar S A , Pacquette M
Published: January 2020

Baths with 2% alkaline glutaraldehyde are often reused for 14 days to decontaminate flexible fiberoptic endoscopes (FFEs) between patients, but the effect of such reuse on the disinfectant's activity has not been known. Many busy endoscopy units also disinfect FFEs with contact times shorter than those recommended by the disinfectant manufacturer. We therefore collected samples of the disinfectant over the 14-day reuse period from two manual and one automatic bath used for bronchoscopes and gastroscopes at a local hospital. Control samples were also collected from a manual bath of 2% alkaline glutaraldehyde which did not receive any endoscopes. The germicidal activities of the samples were assessed in a carrier test against a mixture of hepatitis A virus, poliovirus 1 (Sabin), and Pseudomonas aeruginosa; the mixture also contained either Mycobacterium bovis or Mycobacterium gordonae. Bovine serum (5%) was the organic load. The criterion of efficacy was a minimum of a 3-log10-unit reduction in the infectivity titers of the organisms tested. The initial disinfectant concentration in all the baths was nearly 2.25%; it became about 1.8% in the control bath and fell to approximately 1% in the three test baths after 14 days. No protein was detected in the control bath, while its concentration rose gradually in the test baths to a maximum of 1,267 micrograms/ml after 14 days. With a contact time of 10 min at 20 +/- 2 degrees C, all the samples from the control bath were effective against all the test organisms and all the samples from all the test baths were also effective against P. aeruginosa. With a contact time of 10 or 20 min at 20+/-2 degrees C, the virucidal and mycobactericidal activities of the samples from the test baths showed broad-spectrum germicidal activity when the contact time was increased to 45 min and the temperature was raised to 25 degrees C. These findings emphasize the care needed in the disinfection of FFEs, especially in view of the increasing threat of AIDS and the resurgence of tuberculosis.
Diagnosis of Chlamydia trachomatis endocervical infections by a commercial polymerase chain reaction assay
Authors: Bauwens J E , Clark A M , Stamm W E
Published: January 2020

We evaluated a prototype polymerase chain reaction (PCR)-based assay for Chlamydia trachomatis developed by Roche Molecular Systems to detect endocervical infection in women. Of 587 endocervical samples obtained from women attending the Harborview Medical Center sexually transmitted diseases clinic, 58 (10%) were positive for C. trachomatis by cell culture. Compared with culture, the PCR method had a sensitivity of 88% (51 of 58) and a specificity of 99.2% (525 of 529). The positive and negative predictive values were 92.7% (51 of 55) and 98.7% (525 of 532), respectively. After resolution of discrepant results whereby true positives were considered to be either culture-positive patients (58 patients) or culture-negative patients positive upon PCR analysis using both plasmid- and major outer membrane protein-based primers (4 patients), the resolved sensitivities of the PCR and culture were 89 and 93%, respectively. We subsequently performed a second analysis of 362 women, comparing the proposed commercial PCR assay from Roche Molecular Systems with chlamydia cultures. Thirty (8%) women were infected with C. trachomatis. Compared with culture, the assay had a sensitivity of 60% (18 of 30) and a specificity of 99% (328 of 332). Repeat PCR assay done 2 to 5 days later subsequently yielded positive results for 7 of 11 PCR-negative samples from culture-positive women. We conclude that the Roche Molecular Systems PCR assay provides highly specific results compared with culture in a high-risk population of women. Further study is needed, however, to more clearly define the sensitivity of the PCR assay in detecting endocervical C. trachomatis infection in women and to identify factors that may compromise sensitivity.
Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections
Authors: Ho D W , Field P R , Irving W L , Packham D R , Cunningham A L
Published: January 2020

Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.
Parallel comparison of accuracy of API 20E, Vitek GNI, MicroScan Walk/Away Rapid ID, and Becton Dickinson Cobas Micro ID-E/NF for identification of members of the family Enterobacteriaceae and common gram-negative, non-glucose-fermenting bacilli
Authors: O'Hara C M , Tenover F C , Miller J M
Published: January 2020

We compared the API 20E (21 h) (API; bioMérieux Vitek, Hazelwood, Mo.), the Vitek GNI card (4 to 18 h) (Vitek; bioMérieux Vitek), the identification portion of the MicroScan Walk/Away Rapid Neg Combo 3 panel (2 h) (W/A; Baxter Diagnostics, Inc., West Sacramento, Calif.), and the Becton Dickinson Cobas Micro ID-E/NF rotor (21 h) (Cobas; Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), versus conventional biochemicals for their abilities to identify accurately 252 strains of biochemically typical and atypical members of the family Enterobacteriaceae and common non-glucose-fermenting gram-negative bacilli. All strains used were included in the data base of each product. At the end of the initial incubation, 194 (77.0%), 213 (84.5%), 198 (78.6%), and 192 (76.2%) strains were correct to the genus and species levels with the API, Vitek, W/A, and Cobas systems, respectively. After additional biochemical tests were performed, as directed by each manufacturer's protocol, the numbers of strains correctly identified to the genus and species levels were 241 (95.6%), 234 (92.8%), 243 (96.4%), and 230 (91.3%) with the four systems, respectively. The errors were random in all systems, with the exception of two atypical Salmonella enteritidis strains, each of which was misidentified by three systems. After the initial recommended incubation period, both API and Cobas were significantly less accurate than Vitek (Yates' corrected P < 0.05). No significant differences were noted between the results of Vitek and W/A or between the results of API and W/A. After additional tests were completed, Cobas was significantly less accurate than W/A (P < 0.05) but was equal in accuracy to Vitek and API. API, Vitek, and W/A were equal in accuracy after these same additional tests. All four systems were significantly more accurate after additional biochemical testing than after the initial reporting period (194 of 252 versus 241 of 252 for API, 213 of 252 versus 234 of 252 for Vitek, 198 of 252 versus 243 or 252 for W/A, and 192 of 252 versus 230 of 252 for Cobas [P<0.05]).
Genetic similarity and phenotypic diversity of commensal and pathogenic strains of Candida albicans isolated from the oral cavity
Authors: Hellstein J , Vawter-Hugart H , Fotos P , Schmid J , Soll D R
Published: January 2020

Colony phenotype and genetic similarity were assessed within and between groups of commensal and pathogenic strains of Candida albicans collected from the oral cavities of individuals in a single geographical locale. Thirty-eight percent of pathogenic isolates contained predominant or minor variant colony morphologies (other than smooth) when samples from the sites of infection were cultured on plates, while 16% of commensal isolates contained minor variant colony morphologies when samples from the sites of carriage were cultured. The genetic similarities of isolates within and between groups were assessed by DNA fingerprinting by using Southern blot hybridization with the fingerprinting probe Ca3 and analysis with the computer-assisted, automated Dendron system. Both the commensal and the pathogenic groups contained a major cluster of genetically similar C. albicans isolates representing 31 and 33% of the strains in the respective groups. When a combined dendrogram of both commensal and pathogenic isolates was generated, the major clusters of genetically similar isolates in each group mixed into one large cluster. Minor clusters in the individual dendrograms also mixed. These results suggest common clonal origins for commensal and pathogenic strains in the same geographical locale.
Isolation of Mycobacterium avium complex from water in the United States, Finland, Zaire, and Kenya
Authors: von Reyn C F , Waddell R D , Eaton T , Arbeit R D , Maslow J N , Barber T W , Brindle R J , Gilks C F , Lumio J , Lähdevirta J
Published: January 2020

Disseminated infection with organisms of the Mycobacterium avium complex (MAC) is a common complication of AIDS in the United States and other developing countries, but it is rare or absent in sub-Saharan Africa. To assess the comparative likelihood of exposure to MAC in these geographic areas, we used a standard protocol to culture 91 water samples from environmental sites and piped water supply systems in the United States, Finland, Zaire, and Kenya. MAC was isolated from all geographic areas and from 22 of 91 (24%) samples. Isolation rates were 13 of 47 (28%) for environmental samples and 9 of 44 (20%) for water supply samples. Overall isolation rates were 18 of 52 (35%) samples in the United States and Finland, whereas they were 4 of 39 (10%) samples in Zaire and Kenya (P = 0.015). MAC isolation rates from water supply systems were 8 of 25 (32%) samples in the United States and Finland and 1 of 19 (5%) samples in Zaire and Kenya (P = 0.056). MAC was isolated from hospital water in the United States and Finland but not in hospital water in Zaire and Kenya. Serovar determinations showed that six of eight isolates from the United States were serovar 4 or 8. One MAC isolate from Zaire was identified as an "X" mycobacterium. These data suggest that exposure to MAC in water is likely in diverse areas of the world, but that the likelihood of human exposure to the organism in water may be slightly less in sub-Saharan Africa than in developed countries in the Northern Hemisphere.
Virological diagnosis of progressive multifocal leukoencephalopathy: detection of JC virus DNA in cerebrospinal fluid and brain tissue of AIDS patients
Authors: Moret H , Guichard M , Matheron S , Katlama C , Sazdovitch V , Huraux J M , Ingrand D
Published: January 2020

Cerebrospinal fluid (CSF) from 12 AIDS patients with clinical signs consistent with progressive multifocal leukoencephalopathy (PML) was examined by the polymerase chain reaction (PCR) for the presence of JC virus (JCV). A specific JCV target sequence was amplified in the CSF from 9 of the 12 patients and also in brain tissue from all nine JCV-positive patients. The clinical course, neuroimaging features, and, in four cases, histopathology of brain tissue proved that the nine patients had PML. In the other three patients with central nervous system disorders, the JCV genome was undetectable by PCR and Southern blot analysis in CSF and brain tissue. The clinical course and neuroimaging features ruled out PML in these three patients. Five CSF samples and five brain tissue specimens from 10 PML-free AIDS patients with central nervous system disorders were all negative for the JCV genome by PCR and Southern blot analysis. These results show that detection of JCV in CSF by PCR is a good alternative to brain tissue studies for the virological diagnosis of PML in AIDS patients.
Bacterial flora of the sigmoid neovagina
Authors: Toolenaar T A , Freundt I , Wagenvoort J H , Huikeshoven F J , Vogel M , Jeekel H , Drogendijk A C
Published: January 2020

The bacterial microbiota of 15 sigmoid neovaginas, created in patients with congenital vaginal aplasia or male transsexualism, was studied. No specimen was sterile, and only normal inhabitants of the colon were cultured. The total counts of bacteria were lower than those reported for healthy sigmoid colons.
Evidence of multiple taxa within commercially available reference strains of Corynebacterium xerosis
Authors: Coyle M B , Leonard R B , Nowowiejski D J , Malekniazi A , Finn D J
Published: January 2020

Attempts to identify coryneform isolates resembling Corynebacterium xerosis can lead clinical microbiologists to identification schemes with conflicting descriptions which result in confusing C. xerosis with Corynebacterium striatum. For the present study we purchased all available American Type Culture Collection and National Collection of Type Cultures reference cultures of C. xerosis (n = 10) and C. striatum (n = 4) and analyzed them as follows: (i) analysis of biochemical reactions in conventional tests and in the Rapid CORYNE system, (ii) whole-cell fatty acid analysis by using the gas-liquid chromatography research software of Microbial ID, Inc., and (iii) analysis of DNA homology in dot blot hybridizations. Three C. xerosis strains were indistinguishable from the C. striatum strains in whole-cell fatty acid analyses and DNA hybridizations and shared very similar biochemical reactions. The remaining seven strains of C. xerosis clustered into five groups on the basis of fatty acid patterns, DNA hybridizations, and biochemical tests. No reference strain of C. striatum fit the species description in Bergey's Manual of Systematic Bacteriology. The type strains of both C. striatum and C. xerosis fit their respective descriptions by the Centers for Disease Control and Prevention. This study suggests that the 10 commercially available reference strains of C. xerosis represent six different taxa which should be assigned to new species.