Rudi Rossau

A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness

The hepatitis B virus (HBV) genotype was determined in a total of 121 plasma samples collected in France and the US from patients chronically infected with HBV. HBV genotype A was predominant in this collection, appearing in 66 samples (54%), while genotypes B, C, D, E and F occurred in 4 (3%), 14 (12%), 23 (19%), 1 (1%) and 0 (0%) of samples, respectively. However, the genotype of a total of 13 (11%) samples (2 from France, 11 from the US) could not be determined with the methodology used. Sequence analysis, and subsequent phylogenetic analysis of the complete genome and the individual open reading frames, showed that the virus isolate from these samples was 3248 bp long and, phylogenetically, did not cluster with any of the known genotypes. This strain was provisionally called HBV genotype G. Virus isolates that were obtained from geographically separated regions like France and the US were closely related to each other. All virus strains analysed contained some characteristic differences when compared to genotype A: a translational stop codon at aa 2 and 28 of the preCore region; a 36 nt (12 aa) insert in the amino-terminal part of the Core antigen (HBcAg); a 2 aa deletion in the carboxy-terminal part of HBcAg; and a 1 aa deletion in the preS1 open reading frame. The deduced amino acid sequence of HBsAg suggests that this newly discovered genotype G strain belongs to serological group adw2.

Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay

SETTING Multidrug resistant Mycobacterium tuberculosis strains are threatening TB control in the world. Rapid diagnosis of resistance is essential for adequate treatment and optimal control of the disease. OBJECTIVE Evaluation of a new technique (Line Probe Assay, LiPA) for easy and rapid detection of Rifampicin resistance (RMPR) of M. tuberculosis. DESIGN After amplification of the region of the RNA polymerase, involved in RMPR, the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained the presence or absence of RMPR M. tuberculosis can be assessed. 67 clinical samples positive in culture for M. tuberculosis were analyzed with LiPA and results were compared with classical susceptibility testing. RESULTS In vitro drug sensitivity testing identified 46 rifampicin sensitive and 21 resistant strains. In 65 of the 67 specimens LiPA results matched classical testing. In two RMPR cases LiPA showed a sensitive pattern. CONCLUSION In contrast to culture and sensitivity testing, where results take on average 6 weeks, LiPA testing is an easy and rapid (< 48 h) method of detecting RMPR M. tuberculosis in clinical samples. Results correlated in 97% of the samples. In the two RMPR samples with a sensitive LiPA pattern another mechanism of resistance is suspected.

Genotypic relationships and taxonomic localization of unclassified Pseudomonas and Pseudomonas-like strains by deoxyribonucleic acid: ribosomal ribonucleic acid hybridizations

The deoxyribonucleic acid (DNA):ribosomal ribonucleic acid (rRNA) hybridization technique was used to reveal the relationships and taxonomic positions of an additional 83 strains belonging to 43 saprophytic or pathogenic Pseudomonas species and 29 named and unnamed Pseudomonas-like strains. The DNA:rRNA hybrids were characterized by the following two parameters: (i) the temperature at which one-half of the hybrid was eluted and (ii) the percentage of rRNA binding (the amount of rRNA bound per 100 g of filter-fixed DNA). We also used, for a limited number of strains, numerical analysis of carbon assimilation tests to delineate the finer taxonomic relationships of organisms. Of the 83 strains examined, 78 could be definitely assigned either to an rRNA branch or to an rRNA superfamily within the Proteobacteria. Only 25 of our strains belong in the genus Pseudomonas sensu stricto (our Pseudomonas fluorescens rRNA branch). In general, about two-thirds of the named Pseudomonas species have been misclassified and are distributed over at least seven genera all through the Proteobacteria. These organisms need to be reclassified and generically renamed according to their phylogenetic relationships. However, more detailed phenotypic and genotypic studies are necessary before definite nomenclatural proposals can be made. A comprehensive list of the phylogenetic affiliations of the Pseudomonas species is included.

TAXONOMY OF MORAXELLACEAE FAM-NOV, A NEW BACTERIAL FAMILY TO ACCOMMODATE THE GENERA MORAXELLA, ACINETOBACTER, AND PSYCHROBACTER AND RELATED ORGANISMS.

DNA-rRNA hybridization results showed that members of the genus Moraxella, members of the genus Psychrobacter and their relatives, members of the genus Acinetobacter, the false neisseriae, and two misnamed Alysiella strains constitute a separate genotypic cluster. A new family, Moraxellaceae, is proposed to accommodate these organisms. The genus Moraxella is the type genus. Within the Moraxellaceae two main groups can be distinguished. One group includes the Acinetobacter species. The other group can be subdivided in four subgroups consisting of (i) the authentic moraxellae (Moraxella lacunata, Moraxella nonliquefaciens, Moraxella bovis, Moraxella catarrhalis, Moraxella caviae, Moraxella ovis, Moraxella cuniculi, and two misnamed Alysiella strains), (ii) the generically misnamed taxon Moraxella osloensis, (iii) the generically misnamed taxon Moraxella atlantae, and (iv) the generically misnamed taxon Moraxella phenylpyruvica, Psychrobacter immobilis, and allied organisms. The Moraxellaceae cluster belongs to the class Proteobacteria and is a member of superfamily II, which includes the authentic pseudomonads and related organisms. It is not related to the family Neisseriaceae.

Oligella, a New Genus Including Oligella urethralis comb. nov. (Formerly Moraxella urethralis) and Oligella ureolytica sp. nov. (Formerly CDC Group IVe): Relationship to Taylorella equigenitalis and Related Taxa

The taxonomic relationships of Moraxella urethralis, the Centers for Disease Control (CDC) group IVe, Taylorella equigenitalis, and other gram-negative bacteria were studied by deoxyribonucleic acid (DNA)-DNA polyacrylamide gel electrophoresis of cellular proteins, and serological, biochemical, and auxanographic analyses. A high relationship was detected between M. urethralis and the CDC group IVe strains. However, no relationship of M. urethralis and CDC group IVe with genuine Moraxella species was observed. We describe a new genus, Oligella, containing two species: Oligella urethralis (to accommodate Moraxella urethralis) and Oligella ureolytica (to accommodate CDC group IVe strains). The type species is Oligella urethralis, with type strain ATCC 17960T. The type strain of Oligella ureolytica is CDC C379 (ATCC 43534, CCUG 1465, LMG 6519). Oligella is a member of rRNA superfamily III, containing, e.g., the Pseudomonas acidovorans and Pseudomonas solanacearum complexes and Chromobacterium, Janthinobacterium, and Neisseria species. The closest relatives of Oligella species are Taylorella equigenitalis and the Alcaligenaceae family.

Ribosomal Ribonucleic Acid Cistron Similarities and Deoxyribonucleic Acid Homologies of Neisseria , Kingella , Eikenella, Simonsiella, Alysiella, and Centers for Disease Control Groups EF-4 and M-5 in the Emended Family Neisseriaceae

We detected distinct taxonomic relationships among the true Neisseria species, Kingella kingae, Kingella denitrificans, Eikenella corrodens, all Simonsiella species, the type strain of Alysiella filiformis, and members of Centers for Disease Control groups EF-4 and M-5. All these taxa constitute one large separate cluster having high levels of ribosomal ribonucleic acid cistron similarity (thermal denaturation temperature range, 74 to 81°C) in ribosomal ribonucleic acid superfamily III. There are at least four subbranches. We found high deoxyribonucleic acid (DNA)-DNA homology values between Neisseria gonorrhoeae and some other true Neisseria species and within the following species: Simonsiella muelleri, Simonsiella crassa, Simonsiella steedae, Kingella denitrificans, and Eikenella corrodens. All of the members of this large cluster have genome base compositions in the range from 42.8 to 57.7 mol% guanine plus cytosine. The molecular complexities of the genomic DNAs are 2.2 x 109 to 2.7 x 109 for Simonsiella and Alysiella species and 1.4 x 109 to 1.8 x 109 for the other members of this large cluster. We formally propose that this large cluster represents the emended family Neisseriaceae containing the following genera and groups: Neisseria, Kingella (not the generically misnamed Kingella indologenes), Eikenella, Simonsiella, Alysiella (not some misnamed strains), and Centers for Disease Control groups EF-4 and M-5. The genera and subgenera Acinetobacter, Moraxella, Branhamella, Psychrobacter, the false neisseriae, and Kingella indologenes should be removed from the Neisseriaceae, as they belong to superfamilies I and II.

Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations

The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.

Gene polymorphisms of Toll-like and related recognition receptors in relation to the vaginal carriage of Gardnerella vaginalis and Atopobium vaginae.

Host genetic factors have previously been found to act as determinants of differential susceptibility to major infectious diseases. It is less clear whether such polymorphisms may also impose on pathogen recognition in mucosal overgrowth conditions such as bacterial vaginosis, an anaerobic overgrowth condition characterised by the presence of a vaginal biofilm consisting of the Gram-positive anaerobes Gardnerella vaginalis and Atopobium vaginae. We selected 34 single nucleotide polymorphisms pertaining to 9 genes involved with Toll-like receptor-mediated pathogen recognition and/or regulation (LBP, CD14, TLR1, TLR2, TLR4, TLR6, MD2, CARD15 and SIGIRR) and assessed in a nested case-control study their putative association with bacterial vaginosis, as diagnosed by Gram staining, and with the vaginal carriage of A. vaginae and G. vaginalis, as determined by species-specific PCR, among 144 pregnant women. Carriage of G. vaginalis during early pregnancy was associated with the -1155A>G substitution in the promoter region of the MD2 gene (p=0.041). The presence of A. vaginae during the first half of the pregnancy was significantly associated with the CD14 intron 2 1342G>T (p=0.039), the TLR1 exon 4 743A>G (p=0.038), and the CARD15 exon 4 14772A>T (p=0.012) polymorphisms, and marginally significantly associated with the LBP exon13 26842C>T (p=0.056), the CD14 promoter -260C>T (p=0.052), and the TLR1 promoter -7202A>G (p=0.062) polymorphisms. However, no association between gene polymorphisms and bacterial vaginosis as such could be documented. Our data suggest that some degree of genetic susceptibility involving pathogen recognition may occur with the key bacterial vaginosis organism, A. vaginae.

The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.

Nucleotide sequence of a 16S ribosomal RNA gene from Neisseria gonorrhoeae.

1 TGAACATAAG AGTTTGATCC TGGCTCAGAT TGAACGCTGG CGGCATGCTT TACACATGCA 61 AGTCGGACGG CAGCACAGGG AAGCTTGCTT CTCGGGTGGC GAGTGGCGAA CGGGTGAGTA 121 ACATATCGGA ACGTACCGGG TAGOGGGGGA TAACTGATCG AAAGATCAGC TAATACCGCA 181 TACGTCTTGA GAGGGAAAGC AGGGGACCTT CGGGCCTTGC GCTATCCGAG CGGCCGATAT 241 CTGATTAGCT GGTTGGCGGG GTAAAGGCCC ACCAAGGCGA CGATCAGTAG CGGGTCTGAG 301 AGGATGATCC GCCACACTGG GACTGAGACA CGGCCCAGAC TCCTACGGGA GGCAGCAGTG 361 GGGAATTTTG GACAATGGGC GCAAGCCTGA TCCAGCCATG CCGCGTGTCT GAAGAAGGCC 421 TTCGGGTTGT AAAGGACTTT TGTCAGGGAA GAAAAGGCTG TTGCCAATAT CGGCGGCCGA 481 TGACGGTACC TGAAGAATAA GCACCGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA 541 GGGTGCGAGC GTTAATCGGA ATTACTGGGC GTAAAGCGGG CGCAGACGGT TACTTAAGCA 601 GGATGTGAAA TCCCCGGGCT CAACCCGGGA ACTGCGTTCT GAACTGGGTG ACTCGAGTGT 661 GTCAGAGGGA GGTGGAATTC CACGTGTAGC AGTGAAATGC GTAGAGATGT GGAGGAATAC 721 CGATGGCGAA GGCAGCCTCC TGGGATAACA CTGACGTTCA TGTCCGAAAG CGTGGGTAGC 781 AAACAGGATT AGATACCCTG GTAGTCCACG CCCTAAACGA TGTCAATTAG CTGTTGGGCA 841 ACTTGATTGC TTGGTAGCGT AGCTAACGCG TGAAATTGAC CGCCTGGGGA GTACGGTCGC 901 AAGATTAAAA CTCAAAGGAA TTGACGGGGA CCCGCACAAG CGGTGGATGA TGTGGATTAA 961 TTCGATGCAA CGCGAAGAAC CTTACCTGGT TTTGACATGT GCGGAATCCT CCGGAGACGG 1021 AGGAGTGCCT TCGGGAGCCG TAACACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTCGTG 1081 AGATGTTGGG TTAAGTCCCG CAACGAGCGC AACCCTTGTC ATTAGTTGCC ATCATTCGGT 1141 TGGGCACTCT AATGAGACTG CCGGTGACAA GCCGGAGGAA GGTGGGGATG ACGTCAAGTC 1201 CTCATGGCCC TTATGACCAG GGCTTCACAC GTCATACAAT GGTCGGTACA GAGGGTAGCC 1261 AAGCCGCGAG GCGGAGCCAA TCTCACAAAA CCGATCGTAG TCCGGATTGC ACTCTGCAAC 1321 TCGAGTGCAT GAAGTCGGAA TCGCTAGTAA TCGCAGGTCA GCATACTGCG GTGAATACGT 1381 TCCCGGGTCT TGTACACACC GCCCGTCACA CCATGGGAGT GGGGGATACC AGAAGTAGGT 1441 AGGGTAACCG CAAGGAGTCC GCTTACCACG GTATGCTTCA TGACTGGGGT GAAGTCGTAA 1501 CAAGGTAGCC GTAGGGGAAC CTGCGGCTGG ATCACCTCCT TTCT