[Calculated antibiotic therapy in gastroenterologic infections]. / Rationale Antibiotikatherapie in der Gastroenterologie.

In gastroenterologic infections a calculated antibiotic therapy is not only determined by the suspected variety of bacterials usually associated with given symptoms but also by considerations of their potential side effects, pharmaokinetics and penetration as well as an assessment of the immune status of the patient. As some antibiotic combinations show synergistic activity whereas others are antagonistic or result in accumulated toxicity, this manuscript presents the antimicrobial profile of clinically important antibiotics including their side effects, kinetics, penetration and possible combinations. From these data recommendations for a rational and calculated antibiotic therapy of gastrointestinal infections are developed taking the guidelines of the respective scientific societies into consideration.

Fast and type-specific analysis of herpes simplex virus types 1 and 2 by rapid PCR and fluorescence melting-curve-analysis.

Diagnosis of central nervous system (CNS) infection with herpes simplex virus (HSV) requires sensitive and rapid techniques. PCR therefore is considered to be the diagnostic gold standard in these cases. However, current PCR protocols are time-consuming and labor-intensive. In addition, the need for post-amplification manipulations increases the risk of laboratory contaminations with amplified products. In order to improve conventional PCR techniques we compared our current semiautomated HSV-PCR-ELISA assay with a new micro-volume rapid-cycle PCR system that combines real-time monitoring and fluorescence melting-curve analysis without the need for post-amplification sample manipulations. Spiking experiments with supernatants of tissue culture-grown HSV type 1 (HSV-1) and type 2 (HSV-2) in HSV-negative control cerebrospinal fluid (CSF) and sterile water revealed that the new rapid cycle PCR protocol is as sensitive and specific as the PCR-ELISA. Furthermore, a mismatch (G:T) within the probe-targeted region of the HSV-2 glycoprotein B gene decreases the probe/product melting temperature (Tm) from 69 degrees C for HSV-1 to 64 degrees C for HSV-2, enabling the simultaneous identification of the two HSV genotypes by melting-curve analysis within one run. This type specificity of the system was confirmed with 30 genital swabs previously analyzed for the presence of HSV-1/2 in cell culture. While our current PCR-ELISA method needs up to 1 day from sample preparation to result generation, the new procedure takes only 1 h. We consider this system as a promising new tool for the analysis of HSV DNA in CSF and in other human body fluids as well as for the diagnosis of other infectious agents where rapid diagnosis, high sensitivity and specificity are required.

Quantification of HIV-1 proviral DNA and analysis of genomic diversity by polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive polymerase chain reaction/temperature gradient gel electrophoresis (PCR/TGGE) protocol was developed for exact quantification of HIV-1 proviral DNA copy numbers in clinical samples. An internal standard (ST) that differs from wildtype-sequences only by a single base exchange was used as a competitor in PCR. Quantification of HIV-1 target sequences was achieved by coamplification of defined copy numbers of ST with wild type target sequences, hybridization of PCR products to a strand-specifically labelled probe, separation of ST and wildtype sequences by TGGE, and determination of the ratio of wildtype and standard sequences by densitometric scanning. Effects of sample preparation, DNA extraction and white blood cell counts were minimized by the additional quantification of beta-globin sequences. With this technique, it was possible to determine precisely the number of HIV target sequences as compared to the number of beta-globin gene copies with a detection limit of two HIV-1 proviral copies. Forty-four peripheral blood mononuclear cell (PBMC) extracts from 39 HIV-1 infected patients were analyzed by PCR/TGGE. HIV-1 proviral DNA levels ranged between 2 and 24190 HIV-copies/10(6) beta-globin copies. In general, patients in the advanced stages of disease and/or with low CD4 counts had much higher proviral DNA levels than patients in early stages or with high CD4 counts. In patients from whom consecutive samples were obtained, progression of disease correlated with a greater than tenfold rise of HIV-copies/10(6) beta-globin copies. Compared to other recently published protocols for proviral DNA quantification, this experimental approach allows in addition direct demonstration of mutations within the amplified region. The competitive PCR/TGGE protocol described in this study is suitable for monitoring fluctuations of proviral DNA levels and to identify the genomic diversity of HIV target sequences simultaneously in one assay.

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild-type (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to 10 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification of beta-globin target sequences and calculation of the ratio of HCMV copies/beta-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/beta-globin ratios (10,000 to 8000 copies HCMV/10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10(6) beta-globin.

[Modern procedures in detecting viral antigens]. / Moderne Verfahren des Virusantigennachweises.

Besides serology and virus isolation, assays for the detection of viral antigens or nucleic acids are of increasing importance in routine diagnosis of viral infectious diseases. Critical steps, however, are the selection of suitable assays and specimens. With respect to clinical diagnosis, novel ultrasensitive methods for in vitro DNA amplification like polymerase chain reaction or ligase chain reaction are of high diagnostic significance e.g. for the demonstration of herpes simplex virus DNA in CSF samples or hepatitis C virus in patient sera.

Cold agglutinins of anti-Pr specificity in rubella embryopathy.

Cold agglutinins of anti-Pr specificity were detected in two newborn infants suffering from serologically ascertained rubella embryopathy, an IgM kappa anti-Pr(a), titer 64, and an IgM lambda anti-Pr1, titer 16. The cases are rare examples of cold agglutinin production in newborns; a possible relationship between anti-Pr specificity and rubella infection is discussed.

Prevalence of antibodies to recombinant virion infectivity factor in the sera of prospectively studied patients with HIV-1 infection.

Two hundred twenty-four sera were collected from 34 HIV-1 infected patients during an observation period of up to 4.5 years (109 patient years of observation). The sera were tested for the presence of antibodies against the HIV-1 virion infectivity factor (vif) protein. Thirty sera from 6 HIV-1 seronegative individuals served as negative controls. The sera were immunoblotted against a recombinant, prokaryotically expressed vif protein. The prevalence of anti-vif antibodies increased significantly with progression of the disease from 18% to 81% (P less than 0.0001) which suggests a possible role of vif in HIV-1 replication and pathogenicity.

The diagnostic significance of the polymerase chain reaction and isoelectric focusing in herpes simplex virus encephalitis.

In this study, serum and CSF samples of 55 neurological patients have been examined to confirm the diagnosis of herpes simplex virus encephalitis (HSVE). Different methods were applied, including serological titer evaluations, determination of intrathecally-produced HSV-specific antibodies by isoelectric focusing with affinity immunoblotting (IEF), as well as HSV-specific ELISA and HSV-specific polymerase chain reaction (PCR). The results of IEF and PCR have been compared and contrasted to develop general directions for virological diagnosis of HSVE. Of 14 patients suffering from clinically diagnosed HSVE, HSVE was confirmed in 12 cases by the demonstration of PCR or IEF positivity. A HSV-specific CNS infection could be excluded in 2 of these 14 patients. In 17 patients suffering from non-HSVE, PCR and IEF results were negative. Twenty-four patients, suffering from other neurological diseases, serving as a control group, were PCR- and HSV-IEF-negative. The study indicated that there are two possibilities for unequivocal demonstration of HSV-specific CNS involvement: first, performance of PCR especially in the acute phase of disease and in suspicious relapses, and second, performance of HSV-specific IEF for determination of intrathecally synthesized HSV-specific antibodies. It is suggested that these two methods should be introduced in routine diagnosis of viral encephalitis.

Antigenic domains of the HIV-1 vif protein as recognized by human sera and murine monoclonal antibodies.

To analyze the vif antibody response in individuals infected with the human immunodeficiency virus type 1 (HIV-1) and to determine antigenic epitopes on the vif protein, 104 HIV-1+ sera were screened for reactivity with a recombinant vif protein; 30 (28.8%) of these sera recognized the recombinant vif protein in immunoblot and were employed, together with 17 HIV-1/vif-negative control sera, in an enzyme immunoassay (EIA)-based epitope scanning assay with 183 overlapping decapeptides that covered the complete amino acid sequence of the HIV-1 vif protein (strain BH10). Of the 30 HIV-1/vif+ sera, 87% reacted with decapeptides comprising the two following epitopes: IEWRKKRY (vif amino acids 87-94) or DRWNKPQ (vif amino acids 172-178). The two epitopes were 89% and 100% conserved among different HIV-1 strains and their antigenicity could be confirmed by computer-assisted predictions of vif antigenic determinants. All the sera reactive with recombinant vif protein and with vif peptides originated from patients in CDC stages III or IV. Two murine anti-vif monoclonal antibodies reacted only with the seven C-terminal amino acids of the vif protein (SHTMNGH), which were not recognized by any of the human sera. Our results may be useful for further studies of vif seroreactivity and for the production of anti-vif mono- or polyclonal antibodies using vif peptides.

In vitro cleavage of HIV-1 vif RNA by a synthetic ribozyme.

Using the method of Haseloff and Gerlach, we constructed a ribozyme specifically targeted against the virion infectivity factor (vif) of HIV-1. Both, the vif gene and an oligonucleotide representing the catalytic RNA sequence were cloned into pSPT19 downstream of the T7 promoter and transcribed with T7 RNA polymerase. Efficient cleavage of vif RNA by the synthetic ribozyme occurred at pH 7.5 and 37 degrees C in the presence of magnesium ions in vitro. No measurable activity was observed with a vif antisense RNA. A deletion in the hybridizing region of the ribozyme decreased the cleavage rate, while a mutation in the consensus cleavage domain abolished its catalytic activity. Thus, we could demonstrate an in-vitro activity of a specifically designed ribozyme against HIV-1 vif RNA.

Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1.

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as beta-galactosidase fusion proteins, covering about 75% of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.

Antibodies to recombinant HIV-1 vif, tat, and nef proteins in human sera.

The prevalence of antibodies against HIV-1 regulatory proteins in sera of HIV-infected patients from different stages of disease was investigated. HIV-1 vif, tat, and nef genes were cloned in procaryotic vectors and were expressed as MS-2 fusion proteins (vif and nef) or as a non-fusion protein (tat). These recombinant proteins were employed in immunoblot experiments. The specificity of the recognition was confirmed by competition experiments and with control sera from HIV-negative patients. Analysis of 136 serum samples revealed a high percentage of antibodies against nef, irrespective of the stage of disease. Antibodies against tat were found less frequently and increased from 16% to 40% with disease progression. Vif antibodies were detected only in a low percentage in early stages of disease, but their prevalence increased to 36% and 72% with progression of disease to AIDS-related complex and AIDS. Our data suggest that the detection of antibodies against nef may represent an additional and useful marker for the diagnosis of HIV infection, whereas the detection of vif antibodies may indicate disease progression.

HSV-1 gB and VZV gp-II crossreactive antibodies in human sera.

The specificity and prevalence of human IgG antibodies crossreactive between HSV-1 (ANG) and VZV (Ellen) was examined in immunoblots. Using antibody fractions purified on HSV- and VZV-coated affinity chromatography columns and by preadsorption of sera with HSV and/or VZV lysates a crossreactivity between HSV-1 gB and VZV gp-II was demonstrated. Crossreaction of human IgG antibodies among other structural and nonstructural viral proteins, however, was not detected. The frequency of human IgG antibodies crossreactive between HSV-1 gB and VZV gp-II was highest in HSV-seropositive patients experiencing an acute primary VZV infection (4 out of 5 sera tested). In contrast, no crossreactive antibodies were found in sera of HSV-seronegative patients with acute primary VZV infection (0/6) or in sera from individuals with acute recurrent HSV or VZV infection (0/12). Analysis of sera from individuals with previous HSV and/or VZV infection showed the presence of antibodies crossreactive between HSV-1 gB and VZV gp-II in 3 out of 30 sera tested.

Replication of herpes simplex virus type 1 in differentiated human promyelocytic HL-60 cells.

The capability of herpes simplex virus type 1 (HSV-1), strain Angelotti (ANG), to replicate in human promyelocytic HL-60 cells treated with 1,2-tetradecanoyl-phorbol-13-acetate (TPA) and dimethyl sulfoxide (DMSO) was examined. Virus titrations and infectious center assays revealed that HSV-1 ANG replicated in nontreated HL-60 cells and in HL-60 cells treated with TPA. An abortive infection was observed in DMSO-stimulated HL-60 cells. Viral DNA synthesis was detected in nontreated and TPA-treated cells, but not in DMSO-treated cells. Analysis of HSV-1 transcripts revealed that albeit the differences in pretreatment, HL-60 cells synthesized viral immediate-early (ICP4) and early (tk and pol) RNAs, whereas a late viral transcript (gC) was almost exclusively detected in nontreated and TPA-treated HL-60 cells. In line with these observations were the results obtained from studies on viral protein synthesis. The immediate-early protein ICP4 was found in all three cell types. Early (pol), delayed-early (gB), as well as late proteins (VP 5, gC) were identified in nontreated and TPA-treated cells, but only in reduced amounts in DMSO-treated cells. These data suggest a translational block of HSV replication in DMSO-treated HL-60 cells at the level of early gene expression.

Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins.

The specificity and neutralizing activity of antibodies against the major herpes simplex virus type 1 (HSV-1) glycoproteins were tested in serum samples of patients with a history of HSV-1 infection. By preabsorption of sera to preparations of native and denatured HSV-1 proteins, followed by immunoblotting and microneutralization, it was shown that the majority of neutralizing antibodies are directed against denaturation-sensitive epitopes. Furthermore, preabsorption of sera to proteins of viral ts and deletion mutants revealed that antibodies specific for gB, gC, and gE had a low neutralizing activity. These results suggest a major role of anti-gD in neutralization of viral infectivity. In addition, it was shown that antibodies directed against the gB monomer were distinct from antibodies against the gB homodimers. The latter, however, did not reveal any measurable neutralizing activity.

Removal of N-linked carbohydrates decreases the infectivity of herpes simplex virus type 1.

Purified preparations of herpes simplex virus type 1 Angelotti were digested with the exoglycosidases sialidase, beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-mannosidase, and with the endoglycosidases Endo-H and Endo-F. It was found that treatment of virions with Endo-F specifically decreased viral infectivity by a factor of 10. This reduction in titre was not associated with any measurable differences in virus adsorption, suggesting a role of N-linked complex type oligosaccharide chains in penetration. In contrast, a reduction in titre observed upon digestion of virions with exoglycosidases could be attributed to a proteolytic contamination in these enzyme preparations. Treatment of virions with Endo-H, demonstrated to be free of proteolytic contamination, did not reduce viral infectivity. Analysis of endoglycosidase-digested virions by monospecific antibodies and immunoblotting revealed a susceptibility of all four major glycoproteins (gC, gB, gE and gD) to Endo-F, but only gB was susceptible to Endo-H treatment. In contrast, of all the exoglycosidases used only sialidase was found to be active towards native viral glycoproteins. Upon analysis of endoglycosidase-digested virions we could not find any evidence for proteolysis, degradation or altered protein composition of viral envelopes. In contrast, vigorous inhibition of glycoprotein glycosylation by tunicamycin led to the formation of physically intact virions almost completely lacking all major glycoproteins. These data show that digestion of intact virions with glycosidases allows an analysis of the functional relevance of carbohydrate residues without any obvious alterations in the virion glycoprotein composition.

Coexisting anti-I and anti-F1/Gd cold agglutinins in infections by Mycoplasma pneumoniae.

192 sera containing cold agglutinins of apparent anti-I specificity were reinvestigated for concomitant cold agglutinins (CA) against sialic acid-dependent antigens. 35 cases of additional anti-F1 and 3 cases of additional anti-Gd were detected. 53% of cases with coexisting anti-I and anti-F1/Gd CA had a clinical diagnosis of pneumonia, in 39% IgM antibodies against Mycoplasma pneumoniae could be demonstrated. Since F1 and Gd antigens are identical with the structures identified as receptors for M. pneumoniae, the findings support the hypothesis that postinfectious CA are directed against the receptor of the infectious agent.