Detection and characterisation of SCCmec remnants in multiresistant methicillin-susceptible Staphylococcus aureus causing a clonal outbreak in a Swedish county.

The purpose of this study was to investigate if multiresistant methicillin-susceptible Staphylococcus aureus (MR-MSSA) causing a clonal outbreak in Östergötland County, Sweden, were derived from methicillin-resistant S. aureus (MRSA) by carrying remnants of SCCmec, and, if so, to characterise this element. A total of 54 MSSA isolates with concomitant resistance to erythromycin, clindamycin and tobramycin from 49 patients (91% clonally related, spa type t002) were investigated with the BD GeneOhm MRSA assay and real-time polymerase chain reaction (PCR) targeting the SCCmec integration site/SCCmec right extremity junction. DNA sequencing of one isolate representing the MR-MSSA outbreak clone was performed by massive parallel 454 pyrosequencing. All isolates that were part of the clonal outbreak carried SCCmec remnants. The DNA sequencing revealed the carriage of a pseudo-SCC element 12 kb in size, with a genomic organisation identical to an SCCmec type ΙΙ element, except for a 41-kb gap. This study demonstrates the presence of a pseudo-SCC element resembling SCCmec type II among MR-MSSA, suggesting possible derivation from MRSA. The presence of SCCmec remnants should always be considered when SCCmec typing is used for MRSA detection, and may not be suitable in locations with a high prevalence of MR-MSSA, since this might give a high number of false-positive results.

The estimated impact of the CCR-5 delta32 gene deletion on HIV disease progression varies with study design. Oslo HIV Cohort Study Group.

OBJECTIVES: To study the impact of the genotype CCR-5 wild-type +/A32 on the progression rate to AIDS and death, and to discuss sources of bias according to study design. METHODS: A prospective study of 310 HIV-positive subjects with follow-up time from study entry (prevalent cohort), and a prospective study of 105 HIV-positive subjects with well-defined time of HIV seroconversion, with follow-up time from the retrospectively assessed date of HIV seroconversion (retrospective incident cohort). RESULTS: Slower progression to AIDS among subjects with CCR-5 +/delta32 than those with CCR-5 +/+ genotype was estimated in the prevalent cohort (P=0.07, log-rank test). Slower progression to death from any cause was also estimated for subjects with CCR-5 +/delta32 (P < 0.05, log-rank test). No differences in survival after AIDS diagnosis were seen (P=0.89, log-rank test). No differences in the progression rate to AIDS (P=0.82, log-rank test) or death (P=0.78, log-rank test) were estimated in the retrospective incident cohort. CONCLUSIONS: The varying estimates of the impact of CCR-5 genotype on progression to AIDS in this and other studies, may be real and reflect differences in the dependence of HIV on the CCR-5 receptor, or may be due to systematic errors caused by study design. Several methodological difficulties occur when the factor studied, such as CCR-5 genotype, is associated both with the risk of being HIV-infected and the progression of disease.

Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen.

We have developed a new magnetic bead antigen capture enzyme-linked immunoassay for the detection of schistosomal circulating anodic antigen. The assay utilizes IgG1 monoclonal antibody coated monodisperse magnetic beads in microtitre trays fitted to a special magnet. The total test time was found to be 1-2 h, using 0.05 mg beads per well. The lower detection level was 0.7 ng AWA-TCA per ml (approximately 0.07 ng CAA per ml). Validation by sera from uninfected and Schistosoma mansoni infected Africans and Norwegians resulted in an assay specificity of 100% and sensitivity was close to 90% for cases excreting more than 100 eggs per gram faeces. At such clinically relevant levels the inter-assay CV was below 10% and photometric absorbance correlated to antigen levels was nearly linear. There was a significant correlation between the magnetic bead EIA absorbance values and the titres obtained using the previously established ELISA. The new bead assay, however, was easier and less laborious because TCA pretreatment and the titration of positive results were unnecessary.

Intersubtype recombinant HIV type 1 involving HIV-MAL-like and subtype H-like sequence in four Norwegian cases.

Suspected epidemiological links between three cases of human immunodeficiency virus type 1 (HIV-1) infection were verified by the finding of a shared unique virus genotype. A probable male index case was not available for testing. Case 1 was a female sexual partner of the index case. Case 2 was an adult son of case 1. Case 3 was a female sexual partner of case 2. The link to the index case was substantiated by the subsequent finding of another female sexual contact of the index case, harboring the same HIV-1 genotype as the three other cases. To characterize the genotype further, the complete provirus nucleotide sequence was obtained directly from blood cell DNA of case 3. HIV cultivated from case 3 demonstrated CCR5 dependence, an extreme slow-low phenotype, and some genotypic features not present in its directly sequenced counterpart. Most of the gag, pol, and vif genes of these viruses clustered with one of the earliest African HIV-1 strains, MAL, previously classified as a recombinant between the subtypes A, D, and I. Most of the rest of the genome was related to subtype H, albeit with less than 90% identity in most regions. These viruses are the only ones shown to display extensive similarity with MAL in the gag-pol region and among the first HIV-1 recombinants described involving subtype H. We postulate that the gag-pol genes of MAL and these viruses are derived from a common ancestor that is not necessarily intersubtype recombinant in the pol region.

Molecular epidemiology of viral infections. How sequence information helps us understand the evolution and dissemination of viruses.

Viruses evolve much faster than cellular organisms. Together with recent advances in nucleic acid sequencing and biocomputing, this allows us to distinguish between related strains of viruses, and to deduce the relationships between viruses from different outbreaks or individual patients. Databases of nucleotide sequences contain a large number of viral sequences with which novel sequences from local outbreaks can be compared. In this way the dissemination of viruses can be followed both locally and globally. We here review the biological and technological background to the use of virus nucleic acid sequences in epidemiological studies, and provide examples of how this information can be used to monitor human viruses. Molecular studies are particularly valuable for understanding the dissemination and evolution of viruses. The knowledge obtained is useful in epidemiological reconstructions, in real-time surveillance, and may even enable us to make predictions about the future developments of viral diseases.

Detection of human astrovirus serotype 1 by the polymerase chain reaction.

Astroviruses are small, plus-strand RNA viruses associated with diarrhoea, mostly in children. The diagnostic method commonly used is electron microscopy. We have designed a nested polymerase chain reaction (PCR) based on the recently reported nucleotide sequence of the 3' end of the genome of a human astrovirus serotype 1, the most common form. The PCR was positive for the ten serotype 1 samples tested, while being negative for all other viruses tested, including astrovirus type 2, 3, 4 and 5, calicivirus, rotavirus and picornaviruses. Fecal extracts from patients with diarrhoea were analysed directly or after isolation of RNA, the former method being at least as sensitive. Titration of fecal extracts by PCR indicated the presence of up to 10(11) viral particles per ml in feces.

Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction.

An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.

Detection of all serotypes of human astrovirus by the polymerase chain reaction.

A reverse transcription (RT) and polymerase chain reaction (PCR) was designed for the detection of astroviruses based on a conserved nucleotide sequence in the 3'-end of the genome of the 7 known serotypes of human astrovirus. Thirty-eight samples found to contain astrovirus by electron microscopy (EM) were used for evaluation of the assay. The samples were dialyzed for 1 h to remove potential low molecular weight inhibitors of the RT-PCR. Immediately before RT, 1 microliters of the samples were incubated at 94 degrees C for 2 min to disrupt the viral particles. Thirty-six of the samples were positive by PCR, including samples of all 7 serotypes. The two samples that were negative, could hve been false positive by EM, or the viral RNA could have been degraded. All other viruses examined, including calici-, rota- and enteroviruses, were negative.

Reliable isolation of human immunodeficiency virus from cultures of naturally infected CD4+ T cells.

A procedure for reliable and reproducible isolation of human immunodeficiency virus (HIV) from cultures of CD4+ T cells from healthy HIV seropositive individuals is described. Using immunomagnetic cell separation techniques, CD4+ T cells were positively selected from blood or peripheral blood mononuclear cells from 34 HIV infected individuals in CDC group II. The cells were stimulated with beads coated with a monoclonal antibody specific for the T cell receptor alpha/beta dimer and cultured in medium containing recombinant IL 2. HIV was isolated from 100% of the 41 cultures from 34 individuals. As this culture system allows reproducible isolation of HIV from cultures of naturally infected CD4+ T cells in the absence of other autologous or allogeneic cells, it may provide a good test system for the study of factors affecting the replication of HIV at low multiplicities of infection.

Quantitative detection of schistosomal circulating anodic antigen by a magnetic bead antigen capture enzyme-linked immunosorbent assay (MBAC-EIA) before and after mass chemotherapy.

The magnetic bead antigen capture enzyme-linked immunosorbent assay (MBAC-EIA) has been applied to detect schistosomal circulating anodic antigen (CAA) in pre- and post-treatment sera from 55 individuals in a Schistosoma mansoni control project in the Blue Nile valley of western Ethiopia. The amounts of CAA detected by this assay were positively correlated with the numbers of eggs per gram of faeces (epg). A significant reduction in CAA levels as measured by the MBAC-EIA was observed after mass chemotherapy. The sensitivity was 88-89% in clinically significant cases excreting more than 100 epg. In light infections, however, the sensitivity was lower. None of 32 uninfected Norwegian blood donors or 12 Ethiopian immigrants to Norway were positive. The specificity was thus estimated to be 100%. The test is rapid (1-2 h) and simple to perform without sophisticated equipment and could therefore, with slight modification, be used as a reliable method of diagnosis at field level in endemic areas undergoing mass chemotherapy campaigns or population surveys.

Rapid MRSA test in exposed persons: costs and savings in hospitals.

OBJECTIVE: To study a rapid Xpert polymerase chain reaction (PCR) method in detecting methicillin-resistant Staphylococcus aureus (MRSA) in patients and healthcare workers (HCW) exposed to MRSA, and to estimate savings associated to isolation or work restriction. METHODS: A test set of four double (one for the growth and one for the rapid test) pre-wet swabs from the nose, throat, hands/wrists and perineum was studied by a growth method and by the Xpert MRSA test. RESULTS: The total correspondence between the growth and the rapid test was 92.8%. The overall sensitivity, specificity, positive and negative predictive values were for the Xpert MRSA test: 87%, 99.6%, 68.5% and 99.9%, and for the growth test: 76%, 100%, 100%, and 99.8%, assuming a prevalence of MRSA of 0.01%. Among the MRSA positive persons, the Xpert and growth tests detected MRSA in 44.6% and 40% of nose samples, respectively, 38.2% and 45.5% throat samples, 30.8% and 11.5% hands/wrists samples, 44% and 38% perineum samples, and in 81.8% and 77.3% wound samples, respectively. By combining four anatomical sites, the detection rate increased to 87.5% by both methods. The cost for each Xpert and growth test was euro50 and euro6.25, respectively. The rapid test would save at least euro925 per exposed HCW and euro550 per patient that were MRSA negative. CONCLUSION: The MRSA Xpert test is easy to perform, has a high negative predictive value, and may be used to control healthcare workers and patients exposed to MRSA. Sampling from multiple anatomical locations is recommended. Still, more then 10% of MRSA positive cases may not be found.

Methionine is a regulator of starvation-induced proteolysis in Tetrahymena.

The ciliate Tetrahymena thermophila responds to starvation by drastically increasing the rate of proteolysis. The response was reversed by resuspending the cells in a defined growth medium. Among the components of this medium only amino acids were active in inhibiting proteolysis. One amino acid, methionine, accounted for at least 75% of the effect of the complete medium, strongly indicating that in Tetrahymena methionine is the main regulator of step-down proteolysis, a process generally connected with autophagy in eukaryotic cells. The fact that one amino acid has such a drastic effect should make the system well suited for further investigations of the regulation of this process.

Proteolytic response to nutritional step-down in Tetrahymena.

The ciliate Tetrahymena thermophila is usually grown in a medium containing proteose peptone and yeast extract as organic nutrients. When the ciliate is transferred to step-down conditions, i.e., an inorganic medium, it is shown that the cells respond by rapidly and drastically increasing their rate of protein degradation. A method for measuring the response to step-down conditions is presented, and the response is characterized. The types of proteinases involved are indicated by the use of specific inhibitors. It is concluded that Tetrahymena reacts in much the same way as mammalian cells, and provides a suitable system for investigating the regulation of protein degradation.

[The HIV proteinase--a target for antiviral agents]. / HIVs proteinase--et mål for antivirale midler.

The enormous resources spent on developing inhibitors of the HIV proteinase is finally proving worth while. The FDA in the USA approved saquinavir (Invirase, Roche) for treatment of AIDS in December 1995, and the presumably even more useful inhibitors, ritonavir (Norvir, Abbott) and indinavir (Crixivan, Merck) in March 1996. The clinical trials indicate that these substances are more efficient antiviral agents than the well known reverse transcriptase inhibitors (e.g., AZT or ddC). In the present article, the function of the HIV proteinase will be discussed, as well as the drug design strategies leading to the success. We believe that the combination of biotechnology and computer modelling is a potent tool for designing drugs, and that these proteinase inhibitors not only signal optimism in the treatment of AIDS, but also a new era in the development of therapeutics.

[Gene therapy. Methods and applications]. / Genterapi. Metoder og anvendelser.

Modern techniques in molecular biology and cell biology will probably make gene therapy, i.e. therapeutic transfer of genes to the patient's cells, available for treatment of many genetic diseases, cancer, cardiovascular diseases and infectious diseases. For genetic diseases the treatment will involve the transfer of a functional copy of the defect gene. The strategy for treatment of cancer may include the transfer of genes that induce the death of cancer cells via toxic molecules, and genes that enhance the immune response to tumour cells. After several years of preclinical studies, the National Institutes of Health in the USA has, up to February 1994, approved 56 protocols for clinical trials in human gene therapy. Most of the protocols include use of viruses to aid gene delivery. This paper briefly reviews the scientific basis for gene therapy, and discusses some clinical applications of somatic gene therapy in humans.

[New cases of HIV infection. Estimation of the time of transmission]. / Nye tilfeller av HIV-infeksjon. Vurdering av tidspunkt for smitte.

During 1991 and 1992, 107 HIV infections were diagnosed at Ullevål hospital, Oslo. Previously collected serum specimens from 53 of these persons were tested with different anti-HIV and HIV antigen tests. Some of the sera were also examined for HIV RNA using PCR. Based on these results, the intervals between the last HIV negative and the first positive samples, and between the first HIV positive sample and the HIV diagnosis, were calculated. 38 persons had delivered HIV negative samples before their first positive sample. In addition, eight persons had a sample with serological markers of recent infection. For these 46 persons, the median time between the last HIV negative sample and the diagnosis was one year. 36 of them were HIV negative three years before diagnosis. For 19 persons, markers of HIV infection were detected in samples taken before the disease was first diagnosed. For two persons HIV markers were present in samples taken as early as 1986.

Molecular characterisation of the 3'-end of the astrovirus genome.

We have sequenced the 3'-end of the RNA genomes of 14 serotyped and 12 untyped isolates of human astrovirus. The sequences, which include all 8 serotypes, were used to predict secondary structures, postulate possible functional domains, reveal conserved regions suitable for nucleic acid amplification and perform phylogenetic analysis. The final nucleotides of the capsid protein precursor gene and the adjacent 3'-noncoding region were highly conserved and, except for 35 nucleotides with homology to a sequence in the 3'-end of a coronavirus RNA genome, unique to astrovirus family. This confirms that the 3'-end is a suitable target for universal and specific detection of astrovirus RNA. For the deduced 72 C-terminal amino acids of the capsid protein precursor, distances between the serotypes were found to vary from 0.1 substitution per site between serotypes 3 and 7 to more than one substitution per site between serotype 4 and the other serotypes. Different isolates of the same serotype were closely related, which indicates that the presently used type-specific antibodies differentiate between phylogenetically distinct groups. RNA secondary structures with minimal free energy were predicted using computer programs. Comparative sequence analysis verified the significance of certain of the predicted structural elements.

A common RNA motif in the 3' end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus.

In the 3' non-coding region of the genomes of infectious bronchitis virus, an avian coronavirus and the picornavirus equine rhinovirus serotype 2, there is a motif with remarkable similarity, both in sequence and folding, to the second RNA stem-loop from the 3' end of the genomes of human astroviruses. This motif was also found in astroviruses of sheep, pig and turkey, suggesting that it is a common feature of all astroviruses. The conserved nature of the motif indicates that there has been strong selection for its preservation. There is significant homology between the regions flanking this motif in infectious bronchitis virus and a continuous RNA sequence at the same distance from the 3' poly(A) tail in some related mammalian coronaviruses. These observations suggest that the presence of the motif in these three viral families is the result of at least two separate RNA recombination events.

Hepatitis C virus (HCV) RNA among anti-HCV-positive blood donors and their recipients.

Seven of 24 blood donors positive in Ortho's first-generation antibody to hepatitis C virus (anti-HCV) test (EIA-1) were also positive in Ortho's second-generation test (EIA-2). All 7 had at least two anti-HCV positive recipients, whereas only 1 of the 17 EIA-2-negative donors had an anti-HCV-positive recipient. This recipient was a multitransfused patient with von Willebrand's disease. Five of the 7 EIA-2-positive donors had detectable HCV RNA. We traced and tested 38 of the still living blood recipients from the 7 EIA-2-positive donors. Twenty-eight of these were EIA-2 positive and 22 were HCV-PCR positive. One patient with Waldenstroem's hypergammaglobulinemia was EIA-2 negative but HCV-PCR positive. All the EIA-2-positive sera showed reactivity in Ortho's recombinant immunoblot assay (RIBA-2), but 5 of the 28 recipients and 1 of the donors reacted with only one band (RIBA-2 indeterminate). Among 32 recipients who probably had received EIA-2-positive blood, 29 (91%) had markers of an HCV infection. Twenty-two (75%) of the HCV-infected recipients had detectable HCV RNA more than 6 months after transfusion and hence were chronic HCV carriers.

Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG.

Substrains of Mycobacterium bovis BCG have been divided in two major groups, high producers and low producers of the secreted proteins MPB64 and MPB70. Of these, Mycobacterium tuberculosis secretes only the analog MPT64 during growth on Sauton medium. It has been confirmed that high-producer and low-producer substrains of BCG as well as M. tuberculosis contain the gene for the MPB/MPT70 protein. By contrast, polymerase chain reaction and hybridization experiments are reported here which indicate that the MPB64 gene is absent in the BCG substrains Copenhagen, Pasteur, Glaxo, and Tice, in which previous methods did not permit distinction between secretion of small amounts or absence of the protein in culture fluids.