# Global Meningococcal Initiative: guidelines for diagnosis and confirmation of invasive meningococcal disease.

J. A. Vazquez, Muhamed-Kheir Taha, J. Findlow, S. Gupta, R. Borrow

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## To cite this version:

J. A. Vazquez, Muhamed-Kheir Taha, J. Findlow, S. Gupta, R. Borrow. Global Meningococcal Initiative: guidelines for diagnosis and confirmation of invasive meningococcal disease. Epidemiology and Infection, 2016, 144 (14), pp.3052-3057. 10.1017/S0950268816001308. pasteur-02060895

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# REVIEW ARTICLE

## Global Meningococcal Initiative: guidelines for diagnosis and confirmation of invasive meningococcal disease

J. A. VÁZQUEZ<fnref n="1" />*†, M. K. TAHA<fnref n="2" />†, J. FINDLOW<fnref n="3" />†, S. GUPTA<fnref n="4" /> AND R. BORROW<fnref n="3" />

Received 11 February 2016; Final revision 26 May 2016; Accepted 1 June 2016;
first published online 30 June 2016

## SUMMARY

The Global Meningococcal Initiative (GMI) is an international group of scientists and clinicians with recognized expertise in meningococcal disease including microbiology, immunology, epidemiology, public health and vaccinology. The GMI was established to promote the global prevention of meningococcal disease through education, research and international cooperation. The GMI held its second summit meeting in 2013 to discuss the different aspects of existing meningococcal immunization programmes and surveillance systems. Laboratory confirmation and characterization were identified as essential for informing evidence-based vaccine implementation decisions. The relative merits of different confirmatory methodologies and their applications in different resource settings were a key component of the discussions. This paper summarizes the salient issues discussed, with special emphasis on the recommendations made and any deficiencies that were identified.

**Key words:** Diagnosis, GMI, meningitis, meningococcal disease.

## INTRODUCTION

The Global Meningococcal Initiative (GMI) is an independent, expert forum of scientists, clinicians and public health officials from across the world. GMI members have recognized expertise in meningococcal disease (MD), including aspects related to microbiology, immunology, epidemiology, public health and vaccinology. The Initiative aims to promote the global prevention of MD through education, research and international cooperation [1].

From data presented at a GMI summit meeting in 2010, it was apparent that meningococcal epidemiology is continuously changing and that there are significant regional and temporal differences. It was therefore recommended that country-/region-specific vaccination recommendations – as opposed to global guidelines – should be developed [1]. Consequently, subsequent GMI meetings have been regionally focused [2, 3], but the GMI has maintained its general principles regarding the promotion of laboratory-based surveillance as the main tool for understanding the true burden of the disease, as well as evaluating meningococcal immunization programmes, particularly for resource-poor countries [1].

The GMI held its second summit meeting in 2013 to discuss various aspects of existing meningococcal

<footnote n="1">¹National Reference Laboratory for Meningococci, Institute of Health Carlos III, Majadahonda Spain</footnote>

<footnote n="2">²Institut Pasteur, Invasive Bacterial Infections Unit and National Reference Centre for Meningococci, Paris, France</footnote>

<footnote n="3">³Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK</footnote>

<footnote n="4">⁴Department of Microbiology, National Centre for Disease Control, New Delhi, India</footnote>

<footnote>* Author for correspondence: J. A. Vázquez, National Reference Laboratory for Meningococci, Institute of Health Carlos III, 28220 Majadahonda (Madrid), Spain.
 (Email: jvazquez@isciii.es)
† These authors contributed equally to this work.</footnote>

<footnote>This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.</footnote>
immunization surveillance programmes, with a focus
on the areas where knowledge and experience are
lacking. The relative merits of different confirmatory
and characterization methodologies, and their appli-
cation into different resource settings, were a key com-
ponent of the discussions. This paper summarizes the
salient issues discussed at this meeting and the subse-
quent discussions.

SAFETY CONSIDERATIONS WHEN
HANDLING MENINGOCOCCI IN THE
LABORATORY

*Neisseria meningitidis* is classified as a hazard group 2 organism and should to be handled under containment level 2 or higher. Laboratory staff will be at risk when handling live meningococcal cultures and clinical samples containing viable meningococci if the appropriate safety controls are not maintained [4].

Laboratory staff

Staff must be informed and trained on the biological
risks of working with meningococci and informed
on the signs and symptoms of MD. It is of paramount
importance that all standard operating procedures and
risk assessments are implemented and followed.
Vaccination of staff with available meningococcal
vaccines is strongly recommended and vaccinations
against other agents (including hepatitis B) should
be kept up to date.

Laboratory

The main safety risk is exposure to aerosols containing viable meningococci. It is therefore of paramount importance that any procedure that could result in the production of aerosols (e.g. handling liquid suspensions) is undertaken with appropriate containment and, where possible, the risk of aerosol production should be engineered out of the processes (by replacing liquid culture with solid culture, for example).

CURRENT DIAGNOSIS, CONFIRMATION
AND CHARACTERIZATION OF INVASIVE
MENINGOCOCCAL DISEASE (IMD)

Although immediate antibiotic treatment should not
be delayed upon suspicion of IMD, diagnosis requires
rapid confirmation to enable reliable antibiotic treat-
ment of patient and chemoprophylaxis among

contacts. This should be followed by characterization
to further inform contact management; for example,
through immunization if the pathogen is found to be
vaccine preventable.

Diagnosis: clinical criteria

Early signs and symptoms are usually unspecific.
Patients may present with headache, neck stiffness,
fever, projectile vomiting and photophobia, also
with lethargy or altered consciousness in more
advanced disease [5]. Presence of a non-blanching
haemorrhagic rash (purpura fulminans) is distinctive
for IMD and usually prompts immediate antibiotic
treatment. Although meningitis and sepsis are the
two most frequent symptoms, other presentations
may also be observed (e.g. arthritis and pericarditis).
For example, the US Centers for Disease Control
and Prevention (CDC) lists clinical purpura fulminans
as the clinical criterion for suspected IMD [6], while
the European Centre for Disease Prevention and
Control (ECDC) clinical criteria include any person
with at least one of the following: meningeal signs,
haemorrhagic rash, septic shock or septic arthritis
[7]. Australian guidance similarly gives the absence
of evidence for other causes of clinical symptoms,
combined with either clinically compatible disease in-
cluding haemorrhagic rash or compatible disease, plus
close contact with a confirmed case within the previ-
ous 60 days [8].

Case confirmation

Meningococci are Gram-negative diplococci and are
found extracellularly or intracellularly in peripheral
mononuclear leukocytes. The initial laboratory
screening of a potential IMD case can incorporate a
number of widely available and well-established la-
boratory evaluations; samples may be taken from
cerebrospinal fluid (CSF), peripheral blood or skin
lesions or, when symptoms indicate, from synovial,
pleural or pericardial fluid [6]. For example, cytology
using CSF or peripheral blood smears, detection of
bacterial polysaccharides, as well as detection of
blood markers such as C-reactive protein, are methods
available in most hospital laboratories and often en-
able differentiation between viral or bacterial
meningitis.

Recommendations for the use of specific methods
for diagnosis and confirmation will depend largely
on the case definition employed. For example, in
some countries, the presence of Gram-negative diplococci in a CSF or blood smear – or from another normally sterile site – is considered confirmatory, while in others, blood smears from skin lesions are also noted as confirmatory [6–8]. Antigen detection by immuno-histochemistry is also considered confirmatory in some countries [6, 7].

### Microbiological methods

The timing of specimen collection is crucial for successful processing of *N. meningitidis* culture [9]. False-negative results may result from early antibiotic treatment and incorrect handling or culturing of samples [9]. Growth is optimal at 35–37 °C in ~5% CO₂, on blood agar or chocolate agar plates [9, 10].

Detection of soluble bacterial polysaccharides in CSF by latex agglutination is widely used to confirm *N. meningitidis* infection and can provide some typing information. It requires no specialized facilities or training (apart of centrifugation and reagents refrigeration), although timing of specimen collection is an issue and has been associated with false negatives; moreover, results are variable when used for direct detection in clinical samples such as CSF, with sensitivities ranging between 32% and 96% [11], particularly in capsular group B cases. Latex agglutination is also very effective in identifying capsular groups A, B, C, W and Y from culture.

*N. meningitidis* meningococci show a positive result in Kovac's oxidase test. This test determines the presence of cytochrome oxidase, i.e. presence of an organism containing cytochrome c as part of its respiratory chain – this could be *N. meningitidis* but also other bacterial organisms. As such, carbohydrate utilization tests are then used to validate the presence of *N. meningitidis* further [10]. Gram staining costs relatively little and interpretation of the test results requires only minimal resources and staff training. It is usually considered to be highly specific, but is of low sensitivity, particularly in cases with low bacterial load or where early antibiotic treatment has been instigated; it must also be performed in a relatively short period after collection, which is not always feasible where hospitals lack 24-h laboratory facilities. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry has also been described for the routine identification of microorganisms; however, improvement of meningococcal identification is required [12].

In cases with clinically suspicious and negative culture (including in close contacts), nasopharyngeal

swabs can be utilized as they can yield meningococci in up to 50% of instances [13] and can be used to identify and characterize the organism [14]. Although the isolation of meningococci from the nasopharynx in a patient is not considered confirmatory, concordance between nasopharyngeal and invasive isolates has been found in 97% of cases [13].

### Rapid diagnostic tests

Rapid testing using dipstick diagnostic tests is now becoming available and preliminary in-field data appear to be promising [15]. Test kits are designed to be used by non-specialized staff with minimal training in basic facilities. Thus far, tests have been developed for capsular groups A, C, W, Y and X [16, 17], and the GMI recommends that tests for additional capsular groups are developed.

### Polymerase chain reaction (PCR) methods

Compared to conventional culture-based methods, PCR-based methods are not at risk from loss of viable organisms through early antibiotic treatment and sample processing [18, 19]. The production of false positives due to contamination, however, should be avoided by strict adherence to procedural guidelines. The general trend has been to include PCR as confirmatory in case definitions globally, although there is still some level of divergence on this point. For example, in the European Union, 25% of cases are confirmed by PCR alone, and in the UK and Ireland, this rises to ~50% [20]; while in the United States, the CDC only recently included PCR as confirmatory in its case definition [6]. There is as yet no international consensus on their use, although PCR methods incorporating a number of targets have been developed (such as *ctrA*, *porA*, *crgA*, 16S rDNA, *sodC* and *cnl*) [21–25].

The GMI noted that either regional/provincial or centralized reference facilities could be suitable for implementing PCR, providing that results are rapidly available (i.e. within hours). However, although the cost of PCR has been decreasing, its introduction in many countries is still hindered by resource constraints and lack of trained staff; to date, many countries still have not implemented PCR. Finally, despite adoption of PCR, culture is still required to recover viable strains for further characterization and to aid vaccine formulation. Hence, the GMI recommends that confirmatory PCR be instituted wherever possible, but without replacing culture or other methods.
This is likely to result in a more detailed understanding of the real burden of disease in many countries.

## Characterization and surveillance

### *Serogrouping*

Cultured isolates should be systematically serogrouped. The ability of reference laboratories to serogroup *N. meningitidis* is important as it enables public health authorities to identify outbreaks that are controllable by vaccination campaigns, recognize which serogroups are the cause of sporadic disease and detect the emergence of new serogroups. However, detecting the emergence of new outbreak strains does require additional characterization [10].

### *PCR and molecular genetic methods*

Although PCR may be unavailable in remote areas or resource-poor settings, shipping samples to a central laboratory with PCR facilities could enable enhanced surveillance compared to microbiological and serogrouping techniques alone.

In recent years, new PCR and DNA sequencing methodologies have enhanced the characterization of *N. meningitidis* strains [26]. Multilocus sequence typing (MLST), involving characterization of short fragments of several microbial genes, has been utilized in numerous laboratories for successful characterization of isolates and particularly in global surveillance [27]. DNA sequencing methodologies have been used for molecular determination of sensitivity to antimicrobials through identification of allelic variants of specific genes and sensitivity/resistance to the corresponding antibiotics [28]. Specific antigen-sequence typing (AST) to characterize the variable regions of *N. meningitidis*, such as the antigen-encoding genes *porA* and *fetA*, can enable rapid investigation and characterization of antigenic variants as an aid for vaccine development or selection [29]. As whole-genome sequencing (WGS) technology becomes more widely available it is likely to supersede MLST and AST, enabling even more detailed strain characterization [26].

### *Levels of characterization*

There was a consensus that characterization of menin-gococcal isolates should begin as soon as a case has been confirmed. Basic and advanced levels of charac-terization can be defined as follows:

### (1) Basic characterization information:

* Capsular group identification (e.g. by dipstick testing, latex agglutination or PCR).

* *PorA* and *fetA* typing data (e.g. by PCR, AST or WGS) [29].

* Antibiotic susceptibility testing, including a minimum of antimicrobial agents to be tested: two of those used in chemoprophylaxis (rifampicin and quinolones) and two for clinical treatment (penicillin and ceftriaxone), with chloramphenicol included where other agents are not readily available.

### (2) Advanced characterization information:

* Vaccine antigen variant definition (*fHbp*, *NHBA* and *NadA*) (e.g. by AST or WGS) [30].

* Molecular population biology – MLST is currently utilized for this, although WGS will most probably become the established method [31].

* Vaccine antigen expression evaluation using Meningococcal Antigen Typing System [32, 33] or flow cytometry [34] or other similar methodology developed in house.

* Antibiotic susceptibility analysis (e.g. by PCR or WGS) for *penA* (penicillin G), *rpoB* (rifampicin) and *gyrA* (ciprofloxacin) [27, 35–37].

Most of the methodologies listed were of significant value when assessing the impact of interventions of conjugate vaccine against capsular group C and subsequently with quadrivalent conjugate vaccines or conjugate vaccine against capsular group A in the African meningitis belt [38]. The GMI therefore considered that it would be of great interest to use such tools to evaluate the impact of new sub-capsular vaccines for the prevention of capsular group B and recommends that a laboratory or regional laboratories with facilities able to undertake these methods be identified.

## SUMMARY AND CONCLUSIONS

Globally, infrastructure and facilities dictate how MD is confirmed and the level of surveillance undertaken. Laboratory confirmation is essential for informing evidence-based vaccine implementation decisions and many techniques are available, including microscopy, latex agglutination and PCR. The sensitivity and specificity of confirmation techniques can be affected by sample type, sampling procedures and antibiotic use. Most of the confirmation techniques provide results that form the basis of surveillance
and are critical in aiding decisions about vaccine inter-
ventions. There was a general agreement, therefore,
that, regardless of the approach employed for confi-
rmation and further characterization, regular partici-
pation in internal-external quality-assurance schemes
was essential to ensure optimal performance of these
methods. Dipstick tests specific for A, C, W, Y and
X capsular groups show promise for rapid diagnosis
and DNA-based technologies, such as WGS, are
likely to gain in importance for surveillance.
Culture, however, remains the gold standard in confi-
rmation and characterization, as it maintains the iso-
late for further characterization.

ACKNOWLEDGEMENTS

The authors thank Dr T. Jacob John and Dr Godwin
Enwere for their review of this manuscript.

Editorial assistance was provided by Shelley Maria Lindley, PhD, at PAREXEL, which was funded by Sanofi Pasteur. This research received no specific grant from any funding agency, commercial or not-for-profit sectors.

DECLARATION OF INTEREST

J.A.V. has acted as a consultant for and received
travel support from GlaxoSmithKline and Sanofi
Pasteur, and has undertaken contract research on
behalf of the National Institute of Health Carlos III,
Madrid, Spain, for GlaxoSmithKline, Merck,
Novartis, Pfizer and Sanofi Pasteur. J.F. and R.B. per-
formed contract research on behalf of Public Health
England for GlaxoSmithKline, Novartis, Pfizer and
Sanofi Pasteur. J.F. has also acted as a consultant
on behalf of Public Health England and received tra-
vel support for meeting attendance and subsistence
from GlaxoSmithKline, Novartis, Pfizer and Sanofi
Pasteur. M.K.T. performs contract research and ex-
pertise on behalf of the Institut Pasteur for Glaxo
SmithKline, Novartis, Pfizer and Sanofi Pasteur. S.G.
has no conflict of interest to declare.

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