Multiple viral infections after haploidentical hematopoietic stem cell transplantation in a child with acute lymphoblastic leukemia.

After allogeneic hematopoietic stem cell transplantation (HSCT), viral infections/reactivations are a frequent complication, sometimes with fatal outcome. Thus, early diagnosis is recommended by screening of whole blood or plasma preparations using highly sensitive molecular techniques that test for the most common viral pathogens, such as Epstein-Barr virus, cytomegalovirus, and adenoviruses (ADVs). Despite this approach, not every reactivation/infection can be adequately detected or excluded, even with highly sensitive polymerase chain reaction. Particularly after toxic treatment, uncommon infections or infections resistant to first-line treatment can occur, even in unusual locations. Herein, we present the case of a child with Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic HSCT who suffered from 5 different viral reactivations/infections, including acyclovir-resistant herpes simplex virus type 1 esophagitis, human herpesvirus 6 encephalitis, rotavirus gastroenteritis, respiratory syncytial virus pneumonia, and ADV esophagitis, despite routinely performed blood examinations for viral pathogens remaining unrevealing at all times.

Validation of biocides against duck hepatitis B virus as a surrogate virus for human hepatitis B virus.

The use of a surrogate virus, namely duck hepatitis B virus (DHBV), has been recommended for testing the virucidal activity of chemical biocides against hepatitis B virus. To date, however, this model has not been recognized as a standard test in European countries, as its laboratory use is associated with considerable difficulties. As previous studies have demonstrated, several alternative procedures may improve the validation of DHBV infection in a cell culture system. Using indirect immunofluorescent antigen staining and the light cycler real-time polymerase chain reaction (PCR) technique, the virucidal activity of peracetic acid (PAA), povidone-iodine (PVP-I) and formaldehyde was tested against DHBV obtained from congenitally infected ducks or prepared from the transfected hepatoma D2 cell line. The results demonstrated that inactivation of DHBV from the D2 cell line was achieved with lower concentrations of the biocides and within shorter exposure time intervals. These lower concentration-exposure time values for DHBV from D2 cells in comparison with DHBV from infected ducks indicated a higher sensitivity of the virus derived from D2 cells. In addition, concentrations of PAA and PVP-I that significantly inactivated DHBV in suspension tests were not able to destroy the viral genome. In conclusion, DHBV from congenitally infected ducks should be used for virucidal testing of chemical biocides against DHBV; DHBV prepared from D2 cells is unsuitable due to its higher sensitivity to biocides. Indirect immunofluorescent staining allows reliable detection of DHBV infectivity, whereas the hepadnavirucidal effect can be evaluated by quantitative PCR.

Alternative methods for validation of cell culture infection with duck hepatitis B virus.

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.

Molecular characterisation of varicella-zoster virus strains in Germany and differentiation from the Oka vaccine strain.

With the introduction of varicella vaccination, surveillance of varicella-zoster virus (VZV) strains occurring in cases of chickenpox or zoster should be considered. Differentiating Oka vaccine strain from wild-type VZV can be achieved only using molecular genotyping. In the present study, the VZV genotype was examined in 53 VZV strains isolated from patients with varicella or zoster and in 73 samples from skin eruptions, cerebrospinal fluid, and throat swabs obtained from patients with VZV infections in Germany. The polymerase chain reaction and restriction fragment length polymorphisms analysis using DNA fragments of the open reading frames 38, 54, 62, and the R5 repeat region were used. Whereas all VZV isolates could be typed, direct genotyping of viral DNA in patients' samples was achieved in 63 of 73 cases (86.3%). The dominant genotype of VZV found in 88.8% of 116 patients had the wild-type pattern PstI(+) BglI(-) R5A followed by the wild-genotype PstI(+) BglI(+) R5A in 6.0%, the wild-genotype PstI(+) BglI(-) R5B in 3.4%, the wild-genotype PstI(+) BglI(-) R5C and the Oka vaccine genotype PstI(-) BglI(+) R5B in 0.9% of patients each. BglI(-) wild-types were found in 90.7% of patients with zoster and in 9.3% of patients with varicella. By contrast, the BglI(+) wild-type was diagnosed in five patients with varicella and in two patients with zoster. In conclusion, VZV strains found in Germany are similar to strains circulating in the United States and the United Kingdom. VZV wild-type strains containing a BglI restriction site in ORF 54 as well as Oka vaccine strains can rarely be detected.

Binding of a N,N'-bisheteryl derivative of dispirotripiperazine to heparan sulfate residues on the cell surface specifically prevents infection of viruses from different families.

N,N'-bisheteryl derivatives of dispirotripiperazine (DSTP) are a novel class of antiviral compounds with some of their representatives very effectively inhibiting the replication of herpes simplex virus type 1 (HSV-1) in cell culture. Using one representative of these compounds, the N,N'-bis(1-oxido[1,2,5]oxadiazolo[3,4-d]pyrimidin-7-yl)-3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (DSTP 27), we here further tried to elucidate the molecular mechanisms responsible for the antiviral activity. The results from plaque reduction assays under a variety of conditions suggest that inhibition of HSV-1 strain Kupka replication by DSTP 27 occurs at the level of viral attachment by blockade of heparan sulfate (HS) structures on the cell surface that are used as viral receptors. In contrast to heparin and pentosan polysulfate, pretreatment of cells with DSTP 27 resulted in efficient inhibition of viral adsorption and replication persisting several hours after removal of the inhibitor. Specific binding of DSTP 27 to heparin was demonstrated in vitro. Titrations of gC-positive and gC-negative pseudorabies virus (PrV) mutants on HS-positive and HS-negative cell lines confirmed that inhibitory action of DSTP 27 is strictly HS dependent. Aside from HSV-1 Kupka and PrV, DSTP 27 efficiently inhibits growth of several HSV-1 and HSV-2 strains, among them aciclovir/foscarnet-resistant strains, human cytomegalovirus, human respiratory syncytial virus, and human immunodeficiency viruses known to attach to the cell surface via HS.

Semiquantitative PCR analysis of Epstein-Barr virus DNA in clinical samples of patients with EBV-associated diseases.

The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase chain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000-40,000 copies in 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10(5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV-induced transverse myelitis with an increased number of EBV-genome copies in peripheral blood mononuclear cells and EBV-positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplantation, as well as for monitoring the response to therapy.

Successful treatment of Epstein-Barr virus-induced transverse myelitis with ganciclovir and cytomegalovirus hyperimmune globulin following unrelated bone marrow transplantation.

We report a patient who developed Epstein-Barr virus (EBV)-induced transverse myelitis 19 months after unrelated bone marrow transplantation (BMT). The disease was diagnosed by physical examination, serologic determinations, EBV-specific polymerase chain reaction in peripheral blood lymphocytes and cerebrospinal fluid, and characteristic magnetic resonance imaging scan of the spine. The patient was treated with ganciclovir and cytomegalovirus (CMV) hyperimmune globulin. He gradually improved and recovered completely within 4 weeks. This case suggests that ganciclovir and CMV hyperimmune globulin appear to be effective for the treatment of EBV-induced transverse myelitis in immunocompromised patients following BMT.

Coxsackievirus B3-induced chronic myocarditis in outbred NMRI mice.

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population. STUDY DESIGN/METHODS: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA. RESULTS: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection. CONCLUSIONS: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.

Improved isolation of Chlamydia trachomatis from a low-prevalence population by using polyethylene glycol.

The effect of polyethylene glycol (PEG) on the isolation of Chlamydia trachomatis was evaluated in our laboratory. Initial range-finding experiments demonstrated that the number of chlamydial inclusion bodies increased with increasing PEG concentrations. However, PEG concentrations above 10.5% became progressively more toxic to the McCoy cell monolayers. When 50 frozen clinical Chlamydia isolates were inoculated onto McCoy cell cultures with and without 7% PEG, the PEG-treated cultures produced three- to fivefold more chlamydial inclusions than cultures without PEG. This enhancement was also observed when 1,144 fresh clinical specimens from a low-prevalence population were tested. With fresh clinical specimens, PEG-treated cultures produced two- to sixfold more inclusions than standard cultures. The addition of 7% PEG to the chlamydial overlay medium significantly increased the number of inclusions in each culture, improved the sensitivity of the culture, and decreased the probability of missing a weakly positive specimen.

[The effect of potential antineoplastic antibiotics and the metal complex compound cisplatin on in vitro phagocytosis]. / Der Einfluss potentiell antineoplastisch wirksamer Antibiotika und der Metallkomplexverbindung Cisplatin auf die in vitro-Phagozytose.

Under standardized conditions the influence of the drugs lambdamycin, violamycin, granatomycin C, daunorubicin and cisplatin on the phagocytosis has been tested. Therapeutical doses of lambdamycin, daunorubicin and cisplatin did not have any obvious impairment on the phagocytosis. Contrarily to this granatomycin C (an isochromanchinon antibiotic) and violamycin (an anthracyclin in high concentrations) significantly depressed the phagocytosis of PMN.

A phagocytosis capacity assay: parallel measurement of the phagocytosis and the intracellular killing in granulocytes and the influence of some substances on these processes.

A radiometric technique is described for the assessment of phagocytosis and killing of viable yeast cells by granulocytes. This technique does not require separation of extra- and intracellular microorganisms. In this method the phagocytes which contain viable yeast cells (Saccharomyces cerevisiae and Candida albicans) were disrupted by Triton X-100, and only the remaining yeast cells were isotope-labelled. The uptake of [75 Se]L-selenomethionine was used to measure the killing ability of phagocytes. This method is recommended to measure the influence of biological and pharmacological agents to ingest and kill leucocytes in vitro. The following substances affected phagocytosis and killing: Granatomycin C (decreased phagocytosis), cis-DDP (no influence), bestatin (stimulation of phagocytosis) and Z 190/69-HCl (oxazole) (stimulation of phagocytosis and killing).

[Results after insertion of apical titanium cones in apicoectomy]. / Ergebnisse nach Insertion konischer apikaler Verschlusstifte aus Titan bei Wurzelspitzenresektion.

In a post-treatment study 50 apicoectomized teeth in 34 patients being treated with apical titanium cones between 1988 and 1990 were registered. After a mean postoperative observation period of 15.3 months, 42 teeth were still in situ and subjected to clinical and radiographical evaluations. Their mobility was measured by the periotest method. A resulting 84% success rate demonstrates the technique to be suitable to safeguard a prolonged lifetime of severe damaged teeth by use of conical pins which guarantee a perfect closing at neoapex.

Heterologous signal peptide processing in fusion interferon synthesis by engineered L-forms of Proteus mirabilis.

A recombinant DNA Proteus mirabilis L-form expression system, LVI (pJS127), was used to synthesize human fusion interferon alpha 1 (f-IFN-alpha 1). In the expression plasmid used, the complete coding sequence of IFN-alpha 1 was linked to the streptococcal speA promoter and the 5' end of the speA structural gene including its signal sequence coding region. LVI (pJS127) was capable of complete secretion into the culture medium of biologically active f-IFN-alpha 1 whose identity was confirmed by immunological and chemical evidence. In particular, bacterial L-forms were for the first time shown to be capable of correct signal peptide processing, as determined by N-terminal sequencing of the secreted f-IFN.

Development of mathematical models for an in vitro-phagocytosis test system.

Phagocytosis tests have been carried out by many authors using different methods under different conditions. The results have been interpreted in different ways, as well sometimes with conflicting notations. In order to get to a more systematic data analysis and to separate intrinsic from methodic influences, the possibility to apply mathematical models to a phagocytosis test has been studied. In agreement with previous experiences that phagocytosis can well be represented by a mathematical treatment as Michaelis-Menten-type enzyme kinetics concerning its initial rate and by an exponential function under in vivo conditions, in vitro-phagocytosis was phenomenologically described as an analogon of an irreversible bimolecular chemical reaction. In this way, rate and capacity of phagocytosis may be quantified separately. On the basis of systematic deviations of the data from this model, modifications have been developed which could be connected with pertinent observations. The design of further experiments from preliminary results on the basis of our models is discussed.

Fluoride uptake in plaque-covered enamel after treatment with the fluoride lacquer Duraphat.

The amount of alkali-soluble and alkali-insoluble fluoride was determined in human enamel after one- and six-hour treatments with Duraphat. The application was carried out on: (1) slightly demineralized enamel covered with artificial plaque, (2) cleaned, slightly demineralized enamel, and (3) sound enamel without pre-treatment. After a single Duraphat treatment lasting six hours, fluoride uptake was higher than after Duraphat treatment for just one hour in all experimental groups. More fluoride was acquired in both slightly demineralized, plaque-covered and slightly demineralized, cleaned enamel than in sound enamel. Plaque significantly hampered the formation of alkali-soluble fluoride precipitation on demineralized enamel, but its influence on the amount of fluoride taken up by the enamel was minor.

Impact of swine influenza vaccine on serum antibody.

Comparison of a 1976 serum survey with one of 1977 has permitted an assessment of the impact of the national swine influenza vaccine program of 1976-1977 on the antibody status of the Michigan population. Prevalence of HI influenza virus antibody in premarital sera collected in 1976 prior to the vaccine program was compared to that in similar sera collected in 1977. Overall prevalence of A/New Jersey antibody (titers greater than or equal to 1:10) in 1976 sera was 22.3%. Little antibody was detected in sera from persons less than 40 years of age and prevalence peaked at age 50. Increased antibody prevalence was found for all age groups in sera collected in 1977 following the vaccine program, and the overall prevalence was 41.6%. Only 3.5% of those under 19 years of age were vaccinated, and post-vaccine prevalence for this group was 10%. This age group, comprising about 30% of the state population, appears to have had least exposure to swine influenza virus, and may be the population segment at greatest risk of infection should strains of this antigenic composition reappear. In contrast, highest prevalence of A/Victoria antibody was found in the 15 to 19 age group, where prevalence was 52%, compared to an overall prevalence at 40%.