Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.

Gastroretentive dosage forms: overview and special case of Helicobacter pylori.

The challenge to develop efficient gastroretentive dosage forms began about 20 years ago, following the discovery of Helicobacter pylori by Warren and Marshall. In order to understand the real difficulty of increasing the gastric residence time of a dosage form, we have first summarized the important physiologic parameters, which act upon the gastric residence time. Afterwards, we have reviewed the different drug delivery systems designed until now, i.e. high-density, intragastric floating, expandable, superporous hydrogel, mucoadhesive and magnetic systems. Finally, we have focused on gastroretentive dosage forms especially designed against H. pylori, including specific targeting systems against this bacterium.

Comparison of LightCycler PCR and culture for detection of group B streptococci from vaginal swabs.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.

Absence of clonality of Campylobacter jejuni in serotypes other than HS:19 associated with Guillain-Barré syndrome and gastroenteritis.

Guillain-Barré syndrome (GBS) is recognized as a complication that occurs after Campylobacter infection. Certain Penner serotypes, such as HS:19, are linked particularly to GBS in some parts of the world, and there is good evidence for restricted genetic diversity in these isolates. However, GBS also occurs after Campylobacter infection due to other serotypes. Therefore, we asked whether Campylobacter jejuni non-HS:19 serotypes associated with GBS have a clonal structure and differ from strains isolated from patients with Campylobacter gastroenteritis. A worldwide selected population of C. jejuni non-HS:19 strains associated with GBS and gastroenteritis was analyzed by use of multilocus enzyme electrophoresis, automated ribotyping, pulsed-field gel electrophoresis, and flagellin gene typing. The results show that these isolates represent a heterogenic population and do not constitute a unique population across serotypes. No epidemiologic marker for GBS-associated strains was identified.

Molecular population genetic analysis of Campylobacter jejuni HS:19 associated with Guillain-Barré syndrome and gastroenteritis.

Infection with Campylobacter jejuni serotype HS:19 is associated with the development of Guillain-Barré syndrome (GBS). To determine whether a particular HS:19 clone is associated with GBS, multilocus enzyme electrophoresis (MLEE) was used to analyze a worldwide collection of isolates. There were 34 electropherotypes (ETs) in 3 phylogenetic clusters among 83 C. jejuni isolates. Cluster I contained all HS:19 strains, and a single ET (ET4) accounted for most HS:19 strains. HS:19 strains did not occur in any of the other clusters. ET4 contained isolates from different geographic locations, indicating global spread of this clone. Furthermore, ET4 contained isolates from patients with uncomplicated enteritis and GBS, as well as isolates from animal sources. The results of this study show that HS:19 strains comprise a clonal, although not monomorphic, population, which is distinct from non-HS:19 strains within C. jejuni. A unique clone associated with GBS was not identified by use of MLEE.

Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study.

BACKGROUND: Hepatitis C virus (HCV) infection is highly prevalent among HIV-1-infected individuals, but its contribution to the morbidity and mortality of coinfected patients who receive potent antiretroviral therapy is controversial. We used data from the ongoing Swiss HIV Cohort Study to analyse clinical progression of HIV-1, and the virological and immunological response to potent antiretroviral therapy in HIV-1-infected patients with or without concurrent HCV infection. METHODS: We analysed prospective data on survival, clinical disease progression, suppression of HIV-1 replication, CD4-cell recovery, and frequency of changes in antiretroviral therapy according to HCV status in 3111 patients starting potent antiretroviral therapy. RESULTS: 1157 patients (37.2%) were coinfected with HCV, 1015 of whom (87.7%) had a history of intravenous drug use. In multivariate Cox's regression, the probability of progression to a new AIDS-defining clinical event or to death was independently associated with HCV seropositivity (hazard ratio 1.7 [95% CI 1.26-2.30]), and with active intravenous drug use (1.38 [1.02-1.88]). Virological response to antiretroviral therapy and the probability of treatment change were not associated with HCV serostatus. In contrast, HCV seropositivity was associated with a smaller CD4-cell recovery (hazard ratio for a CD4-cell count increase of at least 50 cells/microL=0.79 [0.72-0.87]). INTERPRETATION: HCV and active intravenous drug use could be important factors in the morbidity and mortality among HIV-1-infected patients, possibly through impaired CD4-cell recovery in HCV seropositive patients receiving potent antiretroviral therapy. These findings are relevant for decisions about optimum timing for HCV treatment in the setting of HIV infection.

Phylogeny of the scathophagidae (Diptera, calyptratae) based on mitochondrial DNA sequences.

The family Scathophagidae constitutes, together with members of the families Muscidae, Fannidae, and Anthomyiidae, the Muscoidea superfamily. The species Scathophaga stercoraria has been used extensively to investigate questions in animal ecology and evolution, particularly as a model system for studies of sperm competition and life history evolution. However, no phylogenetic studies have ever been performed on the Scathophagidae and the relationships within this family remain unclear. This study represents a molecular approach aimed at uncovering the phylogenetic relationships among 61 species representing 22 genera of Scathophagidae. A fragment of the terminal region of the mitochondrial gene COI (subunit I of the cytochrome oxidase gene) was sequenced in scathophagid species covering a wide geographic area, as well as a diverse spectrum of ecological habitats. Several clades grouping different genera and species have been identified, but the resolution power of the COI was insufficient to establish the exact relationships between these clades. The molecular data confirm the existence of a group consisting of the genera Delina, Chylizosoma, and Americina, which could represent the subfamily Delinae. Concerning the controversial position of the genus Phrosia, our data clearly suggest that it should be removed from the Delinae and placed within the genus Cordilura. Monophyly of most genera was confirmed, except for the genus Scathophaga, which should be divided into several different taxa.

Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains.

The rdxA gene of 30 independently isolated Helicobacter pylori strains was sequenced. A comparison of the rdxA sequences revealed a higher percentage of amino acid substitutions in the corresponding protein than in other housekeeping genes. Out of 122 point mutations, 41 were missense and 4 were nonsense. A resistant strain with a nucleotide insertion in the rdxA sequence was also found. With the exception of the point mutations and the insertion generating a stop signal, no particular nucleotide mutation or amino acid substitution could be associated to metronidazole resistance. Moreover, phylogenetic analysis of the 30 nucleotide sequences did not demonstrate specific clusters associated with the resistance phenotype.

Prevalence of Helicobacter pylori and hepatitis C virus infections among non-Hodgkin's lymphoma patients in Southern Switzerland.

BACKGROUND AND OBJECTIVE: Several recent studies have reported a high rate of previous hepatitis C virus (HCV) infection in patients with non-Hodgkin's lymphoma (NHL). However, it appears that there are marked geographical differences in the prevalence of HCV among NHL patients. There is further controversy concerning a possible pathogenetic link between HCV and certain histologic lymphoma subtypes, in particular MALT lymphomas, and it has recently been speculated that HCV might be involved in the multistep process of gastric lymphoma genesis, in addition to the well established role of chronic Helicobacter pylori infection. The aim of this study was to investigate the prevalence of HCV and H. pylori infections in patients with B-cell NHL in Southern Switzerland. DESIGN AND METHODS: One hundred and eighty newly diagnosed HIV-negative B-cell NHL patients, consecutively seen at a referral oncology center in Southern Switzerland between 1990 and 1995 were prospectively studied. A microparticle enzyme immunoassay was used to detect antibodies to HCV. Serologic determination of HCV genotype was done by the Murex method. The quantitative detection of IgG anti-H. pylori was performed by the Biorad GAP test. RESULTS: Infection with HCV was detected in 17/180 patients (9.4%; 95% C.I., 6%-15%). This prevalence is significantly higher than that observed in a large survey of 5424 new blood donors from the same area tested in 1992-97 (0.9%; 95% C.I., 0.7-1.2). Neither histologic subtypes nor specific extranodal presentations of NHL were associated with a higher prevalence of HCV. HCV serotype 2 (corresponding to genotypes 2a-c) was the most common. HCV infection was significantly associated with a shorter progression-free survival at both univariate and multivariate analysis. Anti-Helicobacter antibodies were detected in 81/180 patients (45%; 95% C.I., 38%-53%) and H. pylori infection was significantly associated with the development of primary lymphomas of the stomach. INTERPRETATION AND CONCLUSIONS: A high prevalence of HCV infection was detected in NHL lymphoma patients and was associated with a shorter time to lymphoma progression. HCV infection was not correlated with primary gastric presentation or with MALT-type histology. Our findings further support the key role of H.pylori infection in the pathogenesis of primary gastric lymphoma of MALT-type. The possible role of HCV in the pathogenesis of NHL should be further investigated.

Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin.

Primary cutaneous B-cell lymphomas have been associated with Borrelia burgdorferi, the spirochete responsible for Lyme disease. Recently, cutaneous marginal zone B-cell lymphoma has been proposed as a distinct clinical-pathological entity. We report a case of primary cutaneous marginal zone lymphoma, associated with B burgdorferi infection. Polymerase chain reaction (PCR) amplification of the third complementarity determining region (CDR3) of the immunoglobulin heavy chain gene showed the presence of a monoclonal lymphoproliferation, therefore strengthening the histological diagnosis of a malignant process. B burgdorfer-specific hbb gene sequences were detected by PCR in the lymphoma tissue at diagnosis but not after antibiotic treatment. A nearly complete clinical and histological regression was observed after B burgdorferi eradication, with immunohistochemistry studies showing disappearance of plasma cell differentiation and a marked decline in the number of CD3+ T cells and Ki-67+ cells. Our case confirms the link between B burgdorferi and some cutaneous lymphomas. The disappearance of the microorganism accompanied by the unequivocal decrease of most indicators of active T- and B-cell immune response strongly supported a pathogenetic role for B burgdorferi in sustaining an antigen-driven development and growth of this cutaneous marginal zone lymphoma. Antibiotic therapy (analogous to Helicobacter pylori infection in gastric MALT lymphoma) might be helpful with the aim of averting or at least deferring the indication for more aggressive treatment.

Phylogenetic relationships among muscoidea (Diptera: calyptratae) based on mitochondrial DNA sequences.

The utility of a mitochondrial DNA (mtDNA) fragment of about 1100 bp (including partial COI and COII sequences and tRNALeu) for evolutionary studies in Muscoidea is discussed. The species investigated are Scathophaga stercoraria, Microprosopa pallidicauda and Trichopalpus fraterna (family Scathophagidae), Musca domestica (Muscidae), Lasiomma seminitidum (Anthomyiidae) and Fannia armata (Fanniidae). Comparisons were made with published mtDNA sequences of Drosophila, Anopheles and three Calliphoridae species. The molecular phylogeny obtained here matches the classical morphological taxonomy reasonably well. This varies considerably, however, at different taxonomical levels. At a high taxonomic level, there is a clear separation between the Nematocera and the Brachycera, but the Calyptratae-Acalyptratae division is not always supported. At a lower taxonomic level, all species belonging to the same family are well grouped, but at an intermediate level, within the Calyptratae, it is impossible to clearly separate the Muscoidea and Calliphoridae, preventing a firm conclusion on the phylogenetic relationships among Muscoidea families. The entire COI sequence of S. stercoraria, as well as other mtDNA sequences (including the proximal portions of the COI gene, tRNATrp, tRNACys and tRNATyr genes) in Muscoidea species, are also presented and discussed.

High frequency of non-B subtypes in newly diagnosed HIV-1 infections in Switzerland.

HIV-1 subtypes were determined in newly diagnosed residents of Switzerland. Blood was anonymously collected from patients with a first confirmed positive HIV-1 test result. Viral DNA from the env V3-V5 region was amplified by nested polymerase chain reaction (PCR) and screened for subtype B by heteroduplex mobility assay. All amplicons not identified as B were sequenced. From November 1996 to February 1998, 206 samples were analyzed. Main transmission risks were unprotected heterosexual (55.7%) or homosexual (27.1%) sexual contact or intravenous drug use (12.9%). Subtype B dominated in patients of Swiss, other European, American, or Asian citizenship; particularly high frequencies were found in homosexuals (97%) and drug users (94%). Non-B subtypes including A, C, D, E, F, G, H, a possible B/F recombinant, and a sequence related to J were present in 28.2% (95% confidence interval [CI], 22.9%-35.0%). Non-B were frequent in African citizens (95%), heterosexually infected individuals (44%), and women (43%). Heterosexually infected Swiss males harbored non-B strains in 18% and females in 33%. The results document a change in the epidemiology of newly diagnosed HIV-1 infections in Switzerland: predominance of heterosexual transmission and a high frequency of non-B subtypes.

Arguments against the involvement of Borrelia burgdorferi sensu lato in Alzheimer's disease.

The involvement of spirochaetes, such as the aetiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, in Alzheimer's disease (AD), a common neuropathology, has been proposed by several groups in the past. In our laboratory, brains from 10 AD patients were analysed for the presence of B. burgdorferi sensu lato by both standard and nested PCR techniques based on various target regions, such as the hbb gene (encoding the histone-like protein HBb), the fla gene (flagellin), the rrl-rrf ribosomal intergenic spacer region and the rrs gene (encoding 16S rRNA). In addition, ELISA and Western blot tests for the detection of antibodies against spirochaetal antigens were performed on 27 sera from clinical AD patients. Using these methods, we did not obtain any evidence of the involvement of B. burgdorferi in Alzheimer's disease.

Tick zoonoses in the southern part of Switzerland (Canton Ticino): occurrence of Borrelia burgdorferi sensu lato and Rickettsia sp.

The diversity and the distribution of tick species and their infection rates by the pathogenic micro-organism Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, and Rickettsia sp., were studied in Canton Ticino (the southern part of Switzerland). Ticks specimens collected from animals and humans were classified and analysed for the presence of both pathogens. In particular, PCR analysis was performed for the detection of Borrelia spirochetes in Ixodes ricinus and Ixodes hexagonus, and the hemolymph test was done on Rhipicephalus sanguineus for the detection of Rickettsia sp. PCR assays, performed on 424 of the 989 collected ticks, revealed a low rate of infection (around 2%) of both vectors I. ricinus and I. hexagonus by B. burgdorferi sensu lato. These results are in agreement with the modest number of Lyme borreliosis cases yearly recorded in Ticino. Further, through analysis of DNA sequences, the strains carried by the infected ticks were identified as belonging to the genomic group VS116. The widespread finding of the Mediterranean species Rhipicephalus sanguineus in different locations from July 1994 to October 1995 demonstrates its establishment in Ticino. Of the 210 specimens collected, 70 were analysed and one was infected by Rickettsia sp.

A phylogenetic analysis of Borrelia burgdorferi sensu lato based on sequence information from the hbb gene, coding for a histone-like protein.

We describe a phylogenetic investigation of Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, based on a DNA sequence analysis of the hbb gene, which encodes protein HBb, a member of the family of histone-like proteins. Because of their intimate contact with the DNA molecule, these proteins are believed to be fairly conserved through evolution. In this study we proved that the hbb gene is suitable for phylogenetic inference in the genus Borrelia. The hbb gene, which is 327 bp long and encodes 108 amino acids, was sequenced for 39 strains, including 37 strains of B. burgdorferi sensu lato, 1 strain of Borrelia turicatae, and 1 strain of Borrelia parkeri. Genetic variability was determined at the sequence level by computational analysis. Briefly, 81 substitutions were scored at the DNA level. Only 25 of these substitutions were responsible for amino acid substitutions at the translational level. The signature region for bacterial histone-like proteins was found in hbb. Although variable at the nucleotide level, it was highly conserved at the deduced amino acid level. A phylogenetic tree for the genus Borrelia that was generated from multiple sequence alignments was consistent with previously published data derived from DNA-DNA hybridization and multilocus enzyme electrophoresis analyses. The subdivision of B. burgdorferi sensu lato into five species (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, and "Borrelia andersonii") and at least four genomic groups (groups PotiB2, VS116, CA2, and DN127) was confirmed.

A nested polymerase chain reaction for the detection of Borrelia burgdorferi sensu lato based on a multiple sequence analysis of the hbb gene.

A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.

Analysis of the genetic polymorphism of Borrelia burgdorferi sensu lato by multilocus enzyme electrophoresis.

In recent years, Borrelia burgdorferi sensu lato has been subdivided into three species, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, and a new species restricted to Japan, Borrelia japonica, has been isolated from Ixodes ovatus. In addition, members of several new genomic groups have been found in America and in Europe, suggesting that there are additional genospecies. In order to study the diversity of B. burgdorferi sensu lato, we analyzed 54 isolates, cultured from humans and from different tick species and obtained from diverse geographic areas, including Europe, the United States, Japan, and the People's Republic of China. In order to investigate the genetic relationship between microorganisms that are transmitted by soft ticks and microorganisms that cause Lyme disease, we also included three strains of relapsing fever spirochetes. The method which we used was multilocus enzyme electrophoresis; 12 genetic loci were characterized on the basis of the electrophoretic mobilities of their products, and 50 distinct allele profiles (electrophoretic types) were distinguished. The mean genetic diversity per locus was 0.747. a cluster analysis of a matrix of genetic distances for pairs of electrophoretic types revealed 11 divisions that were separated at genetic distances greater than 0.65. Five of these divisions corresponded to B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica, and the newly proposed species "Borrelia andersonii." Our results also confirmed that there are two additional genomic groups in Europe and at least one additional group in the United States. The relapsing fever spirochetes were were not clearly separated from the spirochetes associated with Lyme disease. In conclusion, we believe that the previously proposed subdivision of B. burgdorferi sensu lato into only four species should be reconsidered.