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Direct and Rapid Identification and Genogrouping of Meningococci and porA Amplification by LightCycler PCR
National Reference Laboratory for Pathogenic Neisseria at the Department of Clinical Microbiology & Immunology, Örebro Medical Centre Hospital, Örebro, Sweden.
National Reference Laboratory for Pathogenic Neisseria at the Department of Clinical Microbiology & Immunology, Örebro Medical Centre Hospital, Örebro, Sweden.
National Reference Laboratory for Pathogenic Neisseria at the Department of Clinical Microbiology & Immunology, Örebro Medical Centre Hospital, Örebro, Sweden.
National Reference Laboratory for Pathogenic Neisseria at the Department of Clinical Microbiology & Immunology, Örebro Medical Centre Hospital, Örebro, Sweden.
2002 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 12, 4531-4535 p.Article in journal (Refereed) Published
Abstract [en]

Invasive meningococcal infections are usually diagnosed by culture. This approach is far from optimal due to, e.g., treatment with precollection antibiotics. Molecular-genetics methods are therefore recognized as important tools for optimal laboratory confirmation of meningococcal infections as well as characterization of meningococci (Mc). The aims of the present study were to develop real-time PCRs for identification and genogrouping (A, B, C, Y, and W-135) of Mc and porA amplification that further can be used for subsequent genosubtyping. In a first run Mc were identified. In a second run they were genogrouped and porA genes were amplified. All the Mc isolates (n = 71) but one and cerebrospinal fluid samples (n = 11) tested gave the expected positive results. The specificity, inter- and intra-assay variations, and effects of different amounts of DNA on the melting temperatures were also explored. The LightCycler PCR system was found to effectively detect and characterize Mc in a few hours. This testing, including the DNA sequencing of the porA gene to reveal the genosubtype, does not take more than a working day, and the results can be compared to those from culturing with phenotypic analysis, which takes at least a few days.

Place, publisher, year, edition, pages
2002. Vol. 40, no 12, 4531-4535 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26553DOI: 10.1128/​JCM.40.12.4531-4535.2002Local ID: 11115OAI: oai:DiVA.org:liu-26553DiVA: diva2:247102
Note
On the day of the defence day the status of this article was a manuscript.Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-20Bibliographically approved
In thesis
1. Genetic characterisation of Meningococci
Open this publication in new window or tab >>Genetic characterisation of Meningococci
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Meningococci (Mc) are one of the major causes of meningitis and septicaemia throughout the world. Non-culture diagnostic methods are recognised as important tools for optimal laboratory confirmation of Me infections, due in part to the increased use of preadmission antibiotics. Non-culture methods were designed in England to detect group B, C, Y and W-135 Mc. To add to the repertoire of genetic methods used for grouping, conventional PCR able to identify Me group A DNA from the cerebrospinal fluid was developed (I). This means that most invasive Me can now be detected and directly grouped even in culture-negative samples. The PCR concept was adapted to the direct and rapid LightCycler PCR, which made it possible to detect and characterise Me (A, B, C, Y, W-135 and porA) within a few hours (II). This can be compared to conventional PCR, including gel electrophoresis, which takes at least a full working day. Genetic characterisation, like genosubtyping of Me, is more widely used because of the increased number of samples that are non-serosubtypable by serological methods. Genosubtyping based on three variable regions within the porA gene gives a complete subtype characterisation, which is important for epidemiological purposes as well as for the design of certain vaccines (III-V). The genosubtyping method is suitable as a first step in an epidemiological investigation, while pulsed-field gel electrophoresis is useful for further investigation of similarity between strains in local outbreaks or strains that are suspected to be epidemiologically related (IV & V).

Antibiotic resistance is more frequent these days and is often correlated to genes located in the chromosome or in a plasmid. ß-lactamase producing Me is not common. However, a few Me harbouring plasmids with a ß-lactamase gene have been isolated in the world. The first full sequence of such a plasmid was published in paper VI. It was shown to be a possible variant of a gonococcal plasmid, pJD5, and may therefore have been picked up from that species. It is of great importance to observe the spread of these Me that harbour plasmids like pAB6, since increased spread may require changes in antibiotic treatment strategies.

Abstract [sv]

Meningokockmeningit och/eller sepsis är infektion på hjärnhinnan (hjärnhinneinflammation) och/eller i blodet orsakad av Neisseria meningitidis bakterier, även kallade meningekocker (Mc). Sjukdomen kan framkalla stor rädsla hos många föräldrar, familjemedlemmar och av vårdpersonal eftersom den ofta utvecklas snabbt, har en hög dödlighet och kan vara svår att skilja från andra febersjukdomar. De som överlever kan ha fått permanenta vävnadsskador och neurologiska problem. Den snabba utvecklingen av sjukdomen hos vanligen friska barn eller ungdomar har resulterat i intensiv forskning inom området. Speciellt gäller det diagnostik och karakteriseringsmetoder, vaccinutveckling, samt vad det är som gör vissa sorter av Mc mer virulenta (sjukdomsframkallande) än andra. Denna avhandling (Papper I-VI) handlar om utvecklingen av genetiska metoder för att både mer fullständigt och snabbare kunna identifiera och karakterisera Mc.

Mc sjukdom diagnostiseras vanligen genom att man odlar fram bakterien, men p.g.a. av det ökande användandet av antibiotika och ev. transportproblem kan många prov bli falskt "negativa". För att identifiera de mest sjukdomsframkallande grupperna (A, B, C, Y och W-135) har genetiska metoder nu utvecklats/vidareutvecklats (Papper I, II). Bakterien delas även in i subtyper, vilka också inverkar på bakteriens virulens. Vanligen görs denna karakterisering från odlade bakterier men man har mer och mer övergått till att utgå från bakteriens DNA (genosubtypning) för att få en fullständig karakterisering (Papper III-V). För epidemiologiska syften kan genesubtypning användas tillsammans med en metod där bakteriens DNA delas upp i småbitar för att få ett s.k. "fingeravtryck" av bakterien (Papper IV-V).

I papper VI presenteras den första DNA sekvensen på en ovanligt förekommande Mc plasmid, innehållande genen för ß-laktamas, vilket gör det möjligt för bakterien att byta ner penicillin-antibiotika och gör den därmed resistent. Plasmiden visade sig vara en variant av en plasmid man hittat hos gonokocker (Ge), en nära släkting till Mc, vilket gör det troligt att plasmiden har överförts från Ge. Blir dessa plasmider vanligare hos Mc skulle en förändring av nuvarande antibiotikabehandling av Mc sjukdom behöva göras.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2001. 79 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 697
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28613 (URN)13768 (Local ID)91-7373-135-8 (ISBN)13768 (Archive number)13768 (OAI)
Public defence
2001-11-16, Wilandersalen, Universitetssjukhuset, Linköping, 10:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-20Bibliographically approved

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