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Genetic variant (genovar) determination of Neisseria gonorrhoeae by pyrosequencing of highly polymorphic segments of the porB gene
National Reference Laboratory for Pathogenic Neisseria, Department of Clinical Microbiology, Örebro University Hospital, Örebro.
National Reference Laboratory for Pathogenic Neisseria, Department of Clinical Microbiology, Örebro University Hospital, Örebro.
Unit for Infectious Disease Control, Department of Clinical Microbiology, Örebro University Hospital, Örebro.
Östergötlands Läns Landsting, LMÖ - Laboratoriemedicin i Östergötland.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in the pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The DNA sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy-sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each one of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface exposed amino acid loops of the mature porB protein) ranged from 5 to 11 and 8 to 39, respectively. Among porB1a isolates (n=22) and porB1b isolates (n=65), 22 and 64 unique genovars were identified. All isolates were typeable. The current results provide evidence of a high discriminatory ability, practically the same as sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84322OAI: oai:DiVA.org:liu-84322DiVA: diva2:558727
Available from: 2012-10-04 Created: 2012-10-04 Last updated: 2012-10-04Bibliographically approved
In thesis
1. Genotypic and phenotypic characterisation of Neisseria gonorrhoeae
Open this publication in new window or tab >>Genotypic and phenotypic characterisation of Neisseria gonorrhoeae
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The bacterium Neisseria gonorrhoeae (the gonococcus) is the aetiological agent of gonorrhoea, which remains a major sexually transmitted infection/disease (STI/STD) worldwide. The incidence of gonorrhoea was previously high in many countries, Sweden included. The incidence in Sweden culminated in 1970 with 487 cases per 100,000 inhabitants. After that, the incidence declined almost every year until an all-time low of 2.4 was observed in 1996. ln 1997 the incidence of gonorrhoea began to significantly increase in Sweden, due mainly to a larger number of domestic cases of young heterosexuals of both sexes and homosexual men. This observation, in combination with the widespread use of suboptimal methods for characterisation and, in some countries, for diagnosis of the bacterium, as well as the rapid increase of resistance to several antibiotics, has intensified the research in the field of N. gonorrhoeae.

In the present thesis (paper 1), a high prevalence of decreased susceptibility or resistance to several of the traditionally used gonorrhoea antibiotics was identified and correlated to the geographic area of exposure, especially Asia. Nevertheless, effective antibiotics for treating gonorrhoea are at hand. A substantial genetic heterogeneity within identical serological variants (serovars), i.e. intraserovar, as well as interserovar of N. gonorrhoeae strains circulating within the community was revealed, emphasising the importance of using highly discriminative (DNA-based) epidemiological characterisation methods for the bacteria (papers II-V). Effective DNA-based characterisation methods, i.e. pulsed-field gel electrophmesis (PFGE) of genomic DNA digested with rarely cutting restriction endonucleases and porB gene sequencing, were adapted, optimised and evaluated against conventional phenotypic methods, as epidemiological tools on Swedish N. gonorrhoeae isolates. These molecular techniques showed a significantly higher discriminatory ability, reproducibility, and objectivity than the serovar determinations using the Genetic Systems (OS) or the Pharmacia panel (Ph) ofmonoclonal antibodies (MAbs) (papers II & III). According to a comparison of serologic and genetic parB-based typing of N. gonorrhoeae, the precise amino acid residues of porB, critical for the reactivity of several of the GS MAbs, were difficult to identity (paper IV). In papers IV & V, a determination of genetic group (genogroup) and genetic variant (genovar) was developed based on real-time PCR of the entire porB gene with subsequent sequence analysis in real-time by synthesis, i.e. pyrosequencing technology, of short, highly polymorphic porB gene segments. This method provides a rapid, high-throughput, objective, highly discriminative, typeable, portable for interlaboratory comparisons, and reproducible molecular characterisation for N. gonorrhoeae. Genogroup and genovar determination can complement or even replace the internationally established serovar determination in routine use for following the transmission of individual strains in the community and confirming presumed epidemiological connections or discriminating isolates of suspected clusters of gonorrhoea cases.

Abstract [sv]

Bakterien Neisseria gonorrhoeae (gonokocken) orsakar den sexuellt överförbara infektionen gonorré som med eller utan allvarliga komplikationer, som exempelvis infertilitet och utomkvedshavandeskap, är ett globalt folkhälsoproblem. Infektionen var tidigare mycket vanlig i flertalet länder, så även i Sverige där incidensen kulminerade år 1970 varefter den sjönk i stort sett varje år fram till och med år 1996. Från och med 1997 började infektionens incidens öka igen i Sverige, vilket också kunde noteras i flera andra länder. Framförallt orsakades den svenska ökningen av en ökad inhemsk smittspridning bland yngre heterosexuella kvinnor och män samt homosexuella män. En ökad resistens hos bakterien mot flertalet antibiotika är ett känt problem sedan decennier. De senaste åren har en nationell och internationell intensifiering skett av forskningen. Fokus ligger på effektiva diagnostiska tekniker, optimala karakteriseringsmetoder, epidemiologiska studier samt identifiering och övervakning av begynnande eller ökande antibiotikaresistens. Även patogenes, virulens och vaccinutveckling studeras.

I denna avhandlings arbete I identifierades, bland svenska N. gonorrhoeae isolat, en hög prevalens av nedsatt känslighet eller resistens mot flertalet av de traditionella antibiotika för behandling av gonorré. En korrelation mellan nedsatt känslighet/resistens och geografisk smittort, framförallt Asien, kunde fastställas. Flera olika antibiotika för effektiv behandling finns dock fortfarande tillgängliga. I arbete I-V identifierades en stor genetisk heterogenitet inom och mellan olika serologiska varianter (serovarer) av N. gonorrhoeae stammar. Detta belyser behovet av att använda högdiskriminerande (DNA-baserade) metoder för epidemiologisk karakterisering av bakterien. Effektiva DNA-baserade molekylärgenetiska tekniker, som pulsfältgelelektrofores (PFGE) av genomiskt DNA efter klyvning med restriktionsenzym och sekvenseling av porB genen, adapterades, optimerades och evaluerades mot traditionella fenatypiska metoder, som epidemiologiska verktyg för svenska N. gonorrhoeae isolat. Båda metoderna visade en signifikant högre diskriminemnde förmåga, reproducerbarhet och objektivitet än traditionell karakterisering (arbete II & III). En jämförelse mellan serologisk och genetisk porB-baserad typning av N. gonorrhoeae visade på stora svårigheter att komplett identifiera de använda monoklonala antikropparnas antigena epitoper hos porB (arbete IV). I arbete IV & V utvecklades en bestämning av genetisk grupp (genogrupp) och genetisk variant (genovar) baserad på realtids-PCR av hela porB genen med efterföljande sekvensering i realtidsformat, med hjälp av pyrosequencing teknologi, av korta högvariabla segment av porB genen. Metoden är mycket snabb, högdiskriminerande, reproducerbar, objektiv samt har en hög kapacitet. Metoden skulle kunna ersätta alternativt komplettera den bristfalliga men internationellt etablerade och rutinmässigt använda serovarbestämningen av N. gonorrhoeae för att identifiera spridningen av individuella stammar i samhället, karakterisera isolat i misstänkta kluster av gonorréfall och för rutinmässig partnerspårning.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 103 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 828
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26673 (URN)11240 (Local ID)91-7373-517-5 (ISBN)11240 (Archive number)11240 (OAI)
Public defence
2003-12-19, Wilandersalen, Universitetssjukhuset, Örebro, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-04Bibliographically approved

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Unemo, MagnusOlcén, PerFredlund, Hans

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