1.
Hjelmevoll, Stig Ove, et al.
(författare)
A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene
2006
Ingår i: Journal of Molecular Diagnostics. - : Elsevier BV. - 1525-1578 .- 1943-7811. ; 8:5, s. 574-581
Tidskriftsartikel (refereegranskat) abstract
Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.
2.
Thulin Hedberg, Sara
(författare)
Antibiotic susceptibility and resistance in Neisseria meningitidis : phenotypic and genotypic characteristics
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades. In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.
3.
4.
Hadad, Ronza, 1984-, et al.
(författare)
Novel meningococcal 4CMenB vaccine antigens : prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae
2012
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley-Blackwell. - 0903-4641 .- 1600-0463. ; 120:9, s. 750-760
Tidskriftsartikel (refereegranskat) abstract
The first cross-protective Neisseria meningitidis vaccine (focus on serogroup B), the protein-based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome-derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n similar to=similar to 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.095.4% and 60.994.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross-immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined.
5.
Golparian, Daniel, 1984-, et al.
(författare)
Analytical specificity and sensitivity of the novel dual-target GeneProof Neisseria gonorrhoeae PCR kit for detection of N-gonorrhoeae
2015
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley-Blackwell. - 0903-4641 .- 1600-0463. ; 123:11, s. 955-958
Tidskriftsartikel (refereegranskat) abstract
Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual-target GeneProofNeisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non-gonococcal Neisseria and related species (n=144), and gonococci (n=104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non-gonococcal isolates showed a low-level cross-reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.
6.
Hadad, Ronza, 1984-, et al.
(författare)
Protection against genital tract Chlamydia trachomatis infection following intranasal immunization with a novel recombinant MOMP VS2/4 antigen
2016
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - Hoboken, USA : Wiley-Blackwell. - 0903-4641 .- 1600-0463. ; 124, s. 1078-1086
Tidskriftsartikel (refereegranskat) abstract
The asymptomatic nature of most Chlamydia trachomatis infections and the lack of appropriate effects by current prevention and management call for vaccine development. We evaluated a recombinant subunit vaccine candidate based on the major outer membrane protein variable segments 2 and 4 (MOMP VS2/4). To achieve maximal immunogenicity and ease of production and purification, MOMP VS2/4 was constructed by using highly immunogenic sequences of MOMP only, thereby minimizing the presence of hydrophobic regions, and spacing the immunogenic epitopes with a flexible amino acid sequence. A purification tag was also added. The MOMP VS2/4 was given intranasally, with or without intravaginal boost, with cholera toxin (CT) adjuvant to C57BL/6 mice, which were screened for immunogenicity and protection against a live challenge infection with C. trachomatis serovar D. Bacterial shedding, cell-mediated responses, and antibody responses were monitored. Immunized mice exhibited significantly less bacterial shedding and were better protected against infertility as compared to unimmunized control mice. Immunizations stimulated both systemic and local specific antibody (IgG1, IgG2c, and IgA) responses, and primed T cells that produced interferon-c and interleukins 13 and 17 upon challenge with recall antigen. Thus, MOMP VS2/4, in combination with CT adjuvant, stimulated Th1, Th2, and Th17 effector cells, and generated protective immunity associated with less pathology. We regard MOMP VS2/4 as a promising candidate for further development into a mucosal chlamydial vaccine.
7.
Aspholm, Marina, et al.
(författare)
Helicobacter pylori adhesion to carbohydrates
2006
Ingår i: Methods in Enzymology. - 0076-6879 .- 1557-7988. ; 417, s. 293-339
Tidskriftsartikel (refereegranskat) abstract
Adherence of bacterial pathogens to host tissues contributes to colonization and virulence and typically involves specific interactions between bacterial proteins called adhesins and cognate oligosaccharide (glycan) or protein motifs in the host that are used as receptors. A given pathogen may have multiple adhesins, each specific for a different set of receptors and, potentially, with different roles in infection and disease. This chapter provides strategies for identifying and analyzing host glycan receptors and the bacterial adhesins that exploit them as receptors, with particular reference to adherence of the gastric pathogen Helicobacter pylori.
8.
Lindbäck, Emma, et al.
(författare)
Pyrosequencing of the DNA gyrase gene in Neisseria species : effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae
2006
Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 114:12, s. 837-841
Tidskriftsartikel (refereegranskat) abstract
The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
9.
Lindbäck, Emma, et al.
(författare)
Transformation of ciprofloxacin-resistant Neisseria gonorrhoeae gyrA, parE and porB1b genes
2006
Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 28:3, s. 206-211
Tidskriftsartikel (refereegranskat) abstract
In several transformation experiments, we have shown that introduction of an alteration in GyrA at position 95 of a ciprofloxacin-susceptible Neisseria gonorrhoeae strain (minimum inhibitory concentration (MIC) 0.008 mg/L) increases the MIC to 0.064 mg/L. Two alterations (positions 91 and 95) increase the MIC to 0.125-0.25 mg/L. Transformants with ciprofloxacin MICs of 0.5-16 mg/L were obtained from a moderately ciprofloxacin-resistant strain (MIC 0.25 mg/L). These transformants had alterations in the gene for PorB1b and probably other genes. In one transformant, an alteration in ParE was also introduced, which probably contributed to ciprofloxacin resistance. The ciprofloxacin-resistant transformants had the donor porB1b sequence, and most of them also had altered serovars. We conclude that alterations in N. gonorrhoeae PorB1b could be involved in ciprofloxacin resistance.
10.
Olsen, Birgitta, 1952-, et al.
(författare)
Population-based assessment of Mycoplasma genitalium in Vietnam : low prevalence among married women of reproductive age in a rural area
2009
Ingår i: Journal of the European Academy of Dermatology and Venereology. - Amsterdam : Wiley-Blackwell Publishing Inc.. - 0926-9959 .- 1468-3083. ; 23:5, s. 533-537
Tidskriftsartikel (refereegranskat) abstract
Objective To analyse the prevalence of Mycoplasma genitalium infection in a population-based study among married women from a demographic surveillance site in a rural geographical area of Vietnam.Materials and Methods Women, 18-49 years of age, were randomly selected to participate. DNA was isolated from endocervical swabs sampled from 990 participating women. The M. genitalium MgPa adhesion gene was detected using a real-time PCR with TaqMan probe.Results Eight (0.8% [95% confidence interval, 0.25-1.35%]) of the included women were infected with M. genitalium .Two of these positive women reported clinical symptoms. One additional M. genitalium positive but symptom-free woman, however, showed clinical signs of vaginitis. None of the M. genitalium positive women was concomitantly infected with Chlamydia trachomatis , Neisseria gonorrhoeae , syphilis or HIV. Furthermore, there was no obvious association between M. genitalium infectionand vaginal douching, use of intra-uterine device (IUD), or occurrence of bacterial vaginosis, candidiasis, or Trichomonas vaginalis .Conclusions The prevalence of M. genitalium among married women in Vietnam was relatively low. However, more large, well-designed and appropriately performed studies in other population groups including unmarried women and men, and in other geographical areas, rural as well as urban, are crucial in order to extract any evidence-based conclusions regarding the overall prevalence of STIs, including M. genitalium infections, in the Vietnamese society. The present study compiled with such future studies may form the basis for a national sexual health strategy for prevention, diagnosis, and surveillance of STIs, including M. genitalium infections, in Vietnam.
