the resulting hybridization products on a microtitre solid

Journal of Infection (2001) 42, 100103doi:10.1053/jinf.2001.0814, available online at http://www.idealibrary.com on

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nIntroductionThe development of new technologies for the sensitivedetection of pathogenic microorganisms are frequentlydescribed in scientific journals. However, do the perfor-mance characteristics of newly developed techniquesbased upon nucleic acid amplification match up to theirinitial specification following their development in theresearch laboratory and how often do they transfer intoclinical practice? This article will review the developmentand application of nucleic acid amplification techniquesfor the improved detection and characterization ofNeisseria meningitidis and the ways in which these testsimpact to improve the management and control ofmeningococcal infection.

Meningococcal infection is a life-threatening infectionpredominantly in young children and teenagers, and isoften difficult to diagnose. Although there is increasedawareness of the disease both in the medical professionand the community, the overall mortality remains approx-imately 10% in Western Europe and N. America but varieswith age-specific disease.1 Over the period 19891995the number of notifications rose significantly in Englandand Wales, whilst there was no net change in the numberof culture confirmed cases; during 1995 there wereapproximately 388 (21%) notified cases which wereunconfirmed.2 This growing discrepancy between notifiedand laboratory confirmed cases of meningococcal infec-

SCIENCE AND CL

Nucleic Acid Technologies aA. J.

Manchester Public Health Laboratory, Withition is partly due to the growing practice of early anti-biotic treatment, which significantly reduces thelikelihood of obtaining a positive culture due to the lownumbers or non-viability of organisms present in clinicalsamples.3 Culture confirmation of meningococcal menin-gitis has been further reduced due to the growing reluc-tance to perform lumbar punctures on suspected cases.4

Laboratory confirmation of meningococcal infectionrequires the isolation of organisms from one or more nor-mally sterile body sites, primarily CSF and or blood.Isolation from CSF and blood can be achieved for almost90% and 50% of patients, respectively, but is reduced to

0163-4453/01/02010004 $35.00/05% or less following early antibiotic treatment.5 Currentnon-culture methods for the confirmatory diagnosis ofmeningococcal infection include Gram staining of CSF,detection of capsular polysaccharide (by latex agglutina-tion or staphylococcal co-agglutination, enzyme immuno-assay or counter immunoelectrophoresis) and arefrequently not sensitive enough to detect the presence ofN. meningitidis in clinical samples.6 The diagnosis ofmeningococcal infection from needle biopsy of skinlesions provides an additional method of non-cultureconfirmation of meningococcal infection.7 The growingneed for non-culture methods for the confirmation ofmeningococcal infection was partially addressed by theintroduction of serodiagnosis. Optimal serodiagnosisrequires testing of acute and convalescent specimens,thus providing a retrospective confirmation which may becompromised by both the antigenic variation betweenmeningococci and age-related host immunocompetence.8

Nucleic acid amplification using the polymerase chainreaction (PCR) offers a highly specific and sensitive tech-nique for the direct detection of pathogen genomes,9

including N. meningitidis, in clinical specimens.10 Con-firmatory diagnosis using PCR is performed using smallquantities of clinical material, is faster than culture and isrelatively inexpensive. Additional techniques such ashybridization with specific probes, either on membrane fil-ters (Southern blots) or in solution followed by capture of

ICAL PRACTICE

d Meningococcal Infectionox

gton Hospital, Manchester, M20 2LR, U.K.phase and signal amplification by colorimetric end stagedetection (PCR ELISA), have been developed for detectionof meningococcal DNA.11 The PCR process can be modi-fied to increase sensitivity further by the use of nestedPCR.12 Amplified gene fragments can be analysed bydigestion with restriction endonucleases,13 hybridizationwith specific probes such as 16S rDNA,14 or nucleotidesequencing to provide not only diagnostic but epidemio-logical information from the same sample.15

Non-culture confirmation or diagnosis using sensitivenucleic acid detection techniques such as PCR requires

2001 The British Infection Society

Nucleic Acid and Meningococcal Infection 101careful consideration and choice of gene targets for ampli-fication and the deployment of a strategy to provide accu-rate confirmation of positive and negative results. In orderto maximize sensitivity, insertion sequence elementswhich are often present in multiple copies per genomehave been selected for PCR detection of pathogens16

including N. meningitidis.17 However, there have beenreports of false-positive PCR results using IS gene tar-gets16,18 emphasizing the importance of gene target selec-tion and implementation of a suitable confirmatorystrategy. Other gene targets such as the meningococcalctrA gene which encodes a conserved capsular transportprotein19 and sialyltransferase gene are unique tomeningococci20 and therefore completely specific. ATaqman real time ctrA PCR assay was evaluated using theABI 7700 sequence detection system and found to bemore sensitive and specific compared with an IS1106 PCRELISA assay.21 Specimens sent to the MeningococcalReference Unit for England and Wales for meningococcalPCR testing are now screened by ctrA Taqman assay andall positives confirmed by amplification of the siaD genefor serogroup identity of the organism in question.22 PCRassays have been extended to include serogroups Y andW-135.23 This confirmatory strategy for meningococcalPCR testing has been developed in order to provide aPCR-based service for the non-culture confirmationof meningococcal infection, for which a significant pro-portion of specimens remain unconfirmed by other labo-ratory criteria and for which there is large specimenworkload.

The epidemiology of meningococcal infection rangesfrom pandemic serogroup A disease in sub-SaharanAfrica to endemic group B disease in many developedcountries.24 In addition, the spectrum of disease rangesfrom benign meningococcaemia25 to rapidly fatal fulmi-nant disease and from purely meningitic infection to gen-eralized septicaemia with multi-organ involvement.26

This diverse spectrum of infection requires the collectionof a full specimen set in order to maximize the confirma-tion of a diagnosis.27 The disease spectrum of meningo-coccal infection gives rise to varying sensitivities for PCRdetection depending on the particular disease syndrome.In cases of meningococcal meningitis the highest diag-nostic sensitivity will be obtained by culture or testing CSFwhereas blood or serum/plasma may be less sensitive.However, in cases of meningococcal septicaemia, blood orserum/plasma will provide the highest diagnostic sensitiv-ity for culture or non-culture confirmation of meningo-coccal infection. Whole blood or fractionated blood andbuffy coat extraction may provide increased sensitivity formeningococcal septicaemia. Newcombe et al. havedemonstrated the importance of PCR for the detectionand diagnosis of meningococcal septicaemia using wholeblood buffy coat and serum.28 However, the relative sensi-tivities of PCR versus blood culture may give rise to dis-crepancies despite the high sensitivity of PCR, due to thedifference in blood volume tested. The volumes of blood usedfor culture are in the range of 520 ml versus 100200lfor PCR and the final volume of nucleic acid templateextract added to the PCR reaction ranges from 1 to 5l.Non-culture diagnosis of meningococcal infection by PCRmay be further improved by the development of methods forextraction and isolation of DNA from additional clinicalspecimens, including skin scrapings and biopsy material.

The sensitivity and specificity of PCR assays for the diag-nosis or confirmation of meningococcal infection has beenthe subject of several studies compared with the gold stan-dard of culture from blood or CSF. The sensitivity of PCR forthe confirmation of meningococcal infection has beenshown to be greater than blood culture in all studies andsamples taken after 24 h in one study provided the only testto secure a positive result.29 The impact of non-cultureconfirmation on the laboratory ascertainment of meningo-coccal infection has increased from 20%22 at the timewhen PCR was first introduced for national surveillance forEngland and Wales to 42% in 2000 (personal communica-tion, EB Kaczmarski). The number of cases of meningococ-cal infection confirmed by PCR alone is now significantlymore than by culture and demonstrates the potential ofnucleic acid amplification for the detection of infectionwith the ability to provide genome-based identification.

Epidemiological surveillance, including the identifica-tion of important epidemiological characteristics such asserogroup, serotype and serosubtype, is of maximumimportance in the period immediately prior to and follow-ing the implementation of a major intervention strategysuch as the introduction of a new vaccine. Failure todetermine the major serogroups responsible for disease inthe U.K. due to increasing numbers of unconfirmed casesof meningococcal infection would have impaired thedetermination of the serogroup C conjugate vaccines effi-cacy following the recent large-scale vaccination pro-gramme. Furthermore, serogroup identification formeningococcal infection is important for improved man-agement of disease contacts. In the case of group C infec-tion improved contact management can be achieved bytargeted vaccination, and in the case of confirmed groupB infection major cost savings are made from the preven-tion of inappropriate use of group C vaccine. PCR assaysbased upon serogroup specific nucleotide sequence poly-morphisms in the meningococcal sialyltransferase gene(siaD) have been developed that combine the detection ofmeningococcal DNA with identification of serogroups B,C, Y and W 135 infections.23,30

A. J. Fox102Increased strain discrimination is required for the spe-cific identification of outbreaks, which may be difficult toconfirm when one or more culture negative epidemiologi-cally linked cases occur. The development of a PCR assayusing allelic discrimination, based upon sequence variationin the meningococcal porA and porB genes which deter-mine the organisms serosubtype and serotype, respectively,may provide valuable information for outbreak manage-ment.12,31,32 Recently the technique of MultilocusSequence Typing (MLST) has been described for the defini-tive strain identification of a number of important patho-genic bacteria.33 This technique is of major importance inthe improved characterization and subtyping of pathogenicmicroorganisms, in particular N. meningitidis. MLSTinvolves the direct nucleotide sequencing of the PCR ampli-cons for a panel of gene loci.33 Enright et al. have demon-strated the non-culture identification of Streptococcuspneumoniae directly from CSF specimens using MLST,34 andin the case of S. pneumoniae this may inform the likelihoodof infection due to isolates with altered susceptibility topenicillin. The definitive identification of bacterial strains innon-culture confirmed infections is of growing importance,as in the case of meningococcal infection where some 42%of infections are now confirmed by PCR alone.

The poor immunogenicity of the serogroup B capsularpolysaccharide has hindered the development of an effec-tive vaccine against serogroup B organisms and hasfocused research for a suitable vaccine on outer mem-brane proteins. The identification of the meningococcalPorA as a bactericidal target has led to the development ofa multivalent PorA OMP vaccine35 which has undergonephase II clinical trials in a variety of age groups, includingyoung infants. The capacity for frequent genetic exchangeamongst meningococci results in variation in candidatevaccine antigens such as PorA.36,37 The genetic variationin genes encoding outer membrane proteins, such as PorA,requires the application of sophisticated techniques forthe post-vaccine surveillance. PCR amplification allowsthe development of highly sensitive techniques such ashot-start nested PCR which has been described for themeningococcal porA gene.12 The sensitivity described bySaunders et al. allowed the non-culture detection of themeningococcal PorA subtype (P1.7,3) associated with anoutbreak of meningococcal disease in the city of Iquique,Chile. The usefulness of PCR for confirmatory diagnosis ofmeningococcal infection in post-vaccine surveillance wasalso demonstrated by Caugant et al., who combined thesensitivity of PCR with the direct sequencing of the PCRamplicon, an important attribute of PCR-based technolo-gies, to identify the porA sequences in cases of suspectedculture negative cases of meningococcal infection follow-ing a Norwegian serogroup B OMP vaccine trial.38 The widespread introduction of the Hib polysaccharide-protein conjugate vaccines has resulted in N. meningitidisnow being the major cause of bacterial meningitis.However, in older age groups other bacterial pathogensare of greater importance as causes of meningitis and sep-ticaemia.39 Hence the development of PCR assays basedupon the conserved gene targets such as 16S RNA, butwhich also have genus and species specific variation, havebeen used to identify other bacterial agents responsible formeningitis.40,41 The use of eubacterial PCR for genericdetection of bacterial pathogens in clinical material isproblematic due to the conserved 16S rRNA primersequences and endogenous reagent contamination.42

Alternative strategies have been developed, such as thesimultaneous amplification in a single tube of multiplegene targets from different organisms which cause bacte-rial meningitis and septicaemia.43

The transfer of sensitive and accurate PCR assays forthe non-culture confirmation of meningococcal diseasefrom research to routine laboratory, with large volumesample testing, represents a major challenge for the imple-mentation of these new technologies. Evidence that thedevelopment and establishment of nucleic acid amplifica-tion techniques for detection and characterization ofN. meningitidis has impacted in all areas of disease man-agement demonstrates that the technology has trans-ferred successfully from research to clinical practice.

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IntroductionReferences