EBioMedicine 65 (2021) 103274

Contents lists available at ScienceDirect

EBioMedicine
journal homepage: www.elsevier.com/locate/ebiom

Review

Molecular diagnostic assays for the detection of common bacterial

meningitis pathogens: A narrative review
Kanny Dialloa,b, Vitalis F. Feteha,j, Lilian Ibea,j, Martin Antonioc,k, Dominique A. Caugantd,
Mignon du Plessise, Ala-Eddine Deghmanef, Ian M. Feaversa, Katya Fernandezg,
LeAnne M. Foxh, Charlene M.C. Rodriguesa,i, Olivier Ronveauxg, Muhamed-Kheir Tahaf,
Xin Wangh, Angela B. Brueggemannj, Martin C.J. Maidena, Odile B. Harrisona,*
a
Department of Zoology, University of Oxford, South Parks Rd, Oxford OX1 3SY, United Kingdom
b
Centre Suisse de Recherches Scientifiques en Co ^te d’Ivoire, Abidjan, Cote d’Ivoire
c
WHO Collaborating Centre for New Vaccines Surveillance, Medical Research Council Unit The Gambia at London School of Hygiene & Tropical Medicine, Atlantic
Boulevard, Fajara, PO Box 273, Banjul, Gambia
d
WHO Collaborating Center for Reference and Research on Meningococci, Norwegian Institute of Public Health, Oslo N-0213, Norway
e
A division of the National Health Laboratory Service (NHLS), National Institute for Communicable Diseases (NICD), Johannesburg, South Africa
f
Institut Pasteur, 25-28 Rue du Dr Roux, Paris 75015, France
g
WHO Infectious Hazard Management, Geneva, Switzerland
h
National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Division of Bacterial Diseases, Meningitis and Vaccine
Preventable Diseases Branch, United States
i
Department of Paediatric Infectious Diseases, St George's University Hospitals NHS Foundation Trust, London, United Kingdom
j
Nuffield Department of Population Health, Big Data Institute, University of Oxford, Oxford OX3 7LF, United Kingdom
k
Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

A R T I C L E I N F O A B S T R A C T

Article History: Bacterial meningitis is a major global cause of morbidity and mortality. Rapid identification of the aetiological
Received 20 October 2020 agent of meningitis is essential for clinical and public health management and disease prevention given the
Revised 19 February 2021 wide range of pathogens that cause the clinical syndrome and the availability of vaccines that protect against
Accepted 22 February 2021
some, but not all, of these. Since microbiological culture is complex, slow, and often impacted by prior anti-
Available online 12 March 2021
microbial treatment of the patient, molecular diagnostic assays have been developed for bacterial detection.
Distinguishing between meningitis caused by Neisseria meningitidis (meningococcus), Streptococcus pneumo-
Keywords:
niae (pneumococcus), Haemophilus influenzae, and Streptococcus agalactiae and identifying their polysaccha-
Meningitis
Bacteria
ride capsules is especially important. Here, we review methods used in the identification of these bacteria,
Molecular diagnostics providing an up-to-date account of available assays, allowing clinicians and diagnostic laboratories to make
PCR informed decisions about which assays to use.
RT-PCR © 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
LAMP assay (http://creativecommons.org/licenses/by/4.0/)
Whole Genome Sequence data

1. Introduction children, and adolescents (aged 10 19 years) bacterial meningitis

caused by these organisms typically presents with symptoms includ-
Bacterial meningitis, which can be accompanied by sepsis, is an ing headache, fever, photophobia, vomiting, and neck stiffness [2]. In
infection causing significant morbidity and mortality worldwide [1]. newborns (1 to 28 days), infants (up to 12 months) and young chil-
Many pathogens can invade the membranes lining the brain and spi- dren (from 1 to 10 years), the symptoms and signs are non-specific,
nal cord and cause syndromic meningitis; however, the condition including lethargy, poor feeding, vomiting and irritability associated
can become rapidly fatal if untreated when caused by the encapsu- with fever [3]. Rapid, accurate and specific identification of the causa-
lated bacteria Haemophilus influenzae, Neisseria meningitidis (menin- tive organism is necessary to ensure an effective public health
gococcus), Streptococcus pneumoniae (pneumococcus), and response is elicited and appropriate clinical management, such as
Streptococcus agalactiae (group B streptococci, GBS). In adults, older antimicrobial prophylaxis, can be established with or without vacci-
nation of contacts.
The development and implementation of conjugate polysaccha-
* Corresponding author. ride vaccines at the turn of the century transformed public health
E-mail address: [email protected] (O.B. Harrison).

https://doi.org/10.1016/j.ebiom.2021.103274
2352-3964/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
2 K. Diallo et al. / EBioMedicine 65 (2021) 103274

Table 1
Current status of vaccines against H. influenzae, N. meningitidis, S. pneumoniae and S. agalactiae.

Bacterial species Licensed vaccines and targets Vaccines in clinical development References

Haemophilus ActHIB, Sanofi Pasteur Hib (PRP-T) [84]

influenzae Hiberix, GSK Vaccines Hib (PRP-T)
PedvaxHIB, Merck Hib (PRP-OMP)
Combination vaccines:
Pentacel/Pentaxim, Sanofi Pasteur (DTaP, IPV, Hib [PRP-T])
Hexaxim/Hexyon, Sanofi Pasteur (DTaP, IPV, Hib [PRP-T], HBV)
Vaxelis, Sanofi (DTaP, IPV, Hib [PRP-OMP], HBV)
Infanrix Penta, GSK Vaccines (DTaP, IPV, Hib [PRP-T])
Infanrix Hexa, GSK Vaccines (DTaP, IPV, Hib [PRP-T], HBV)
Menitorix, GSK Vaccines (Hib [PRP-T], MenC [PsC-T])
Menhibrix, GSK Vaccines (Hib [PRP-T], MenCY [PsC-T, PsY-T])
Neisseria Polysaccharide vaccines: [1, 85]
meningitidis Menomune, Sanofi Pasteur (PsA, PsC, PsW, PsY)
Mencevax/ACWYVax, Pfizer (PsA, PsC, PsW, PsY)
NmVac4-A/C/Y/W-135, JN-International Medical Corporation
(ACWY)
Polysaccharide-protein conjugate vaccines: Polysaccharide-protein conjugate vaccines:
Menactra, Sanofi Pasteur (PsA-D, PsC-D, PsW-D, PsY-D) NmCV-5, Serum Institute of India
Menveo, GSK Vaccines (PsA-CRM197, PsC-CRM197, PsW-CRM197, (PsA-T, PsC CRM197, PsW-CRM197,
PsT-CRM197) PsX-T, PsY-CRM197)
Mejugate, GSK Vaccines (PsC-CRM197)
Nimenrix, Pfizer (PsA-T, PsC-T, PsW-T, PsY-T)
NeisVac-C, Pfizer (PsC-T)
MenAfriVac, Serum Institute of India (PsA-T)
Menitorix, GSK Vaccines (Hib [PRP-T], MenC [PsC-T])
Menhibrix, GSK Vaccines (Hib [PRP-T], MenCY [PsC-T, PsY-T])
MenQuadfi, Sanofi Pasteur (MenACWY, [PRP-T])
Outer membrane vesicle vaccines:
VA-MENGOC-BC, Finlay Institute Vaccine, Cuba (CU385/83 B:4:
P1.19, 15) (no longer in production)
Protein-based vaccines:
Bexsero (4CMenB), GSK Vaccines (NZ98/254 OMV, FHbp, NadA,
NHPA)
Trumenba, Pfizer (bivalent FHbp)
Streptococcus Polysaccharide vaccine: [86]
pneumoniae PneumovaxÒ 23 - serotypes:
1, 2, 3, 4, 5, 6B, 7F, 8, 9 V, 9 N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F,
19A, 20, 22F, 23F, 33F
Polysaccharide-protein conjugate vaccines: Polysaccharide-protein conjugate vaccines in Phase
SynflorixTM , GSK Vaccines (10-valent) serotypes: 3 clinical trials:
1, 4, 5, 6B, 7F, 9 V, 14, 18C, 19F, 23F V114, MSD (15-valent) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9 V,
Prevenar/PrevnarÒ , Pfizer (13-valent) serotypes: 14, 18C, 19A, 19F, 22F, 23F, and 33F
1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, 23F 20vPnC, Pfizer (20-valent) serotypes: 1, 3, 4, 5, 6A, 6B, 7F,
Pneumosil, Serum Institute of India (10-valent) serotypes: 8, 9 V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F
1, 5, 6A, 6B, 7F, 9 V, 14, 19A, 19F, 23F
Streptococcus Licenced vaccines not yet available Pfizer: Up to 6-valent vaccine recruiting for trials [7]
agalactiae Minervax:
N-terminal domains of the Rib and AlphaC surface protein
vaccines

management of bacterial meningitis in developed countries, by pro- development, no licenced vaccine against GBS was available at the
viding vaccines that elicited individual protection and herd immunity time of writing [7]. Notwithstanding these limitations, the wide-
against three of the most common causes of meningitis: the menin- spread implementation of available conjugate vaccines has resulted
gococcus; the pneumococcus; and H. influenzae (Table 1) [4]. Each in a range of public health benefits including: (i) The global decrease
polysaccharide vaccine, however, generates a highly specific immune
response only to the polysaccharide antigens it contains. In the case Table 2
of H. influenzae, one capsular type known as serotype b (Hib) is more Known capsular polysaccharides among the four major bacterial meningeal
pathogens.
commonly associated with causing disease, and monovalent vaccines
against Hib have been highly successful. However, other serotypes Bacterial species Known capsular types
(such as serotype a) are increasing in North America and broader vac-
Haemophilus influenzae a b, c, d, e, f
cine development may be required [5]. For the meningococcus, six of Neisseria meningitidis A, B, C, E, H, I, K, L, W, X, Y and Z
the twelve capsular groups (known as serogroups A, B, C, W, X, and Y) Streptococcus pneumoniae 1, 2, 3, 4, 5, 6A, 6B, 6Bii (6E), 6C, 6D, 6F, 6 G, 6H,
cause the majority of meningococcal invasive disease. Conjugate vac- 7F, 7A, 7B, 7C, 8, 9A, 9 L, 9 N, 9 V, 10F, 10A, 10B,
10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A,
cines are available for A, C, W, and Y: a serogroup X conjugate vaccine
12B, 13, 14, 15F, 15A, 15B/C, 16F, 16A, 17F, 17A,
is in clinical trials, and alternative protein-based vaccines are avail- 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20A,
able which protect against a number of meningococci including some 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B,
that express serogroup B capsules [6]. 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F,
For the pneumococcus the challenge is greater, since there are a 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C,
36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46,
large number of capsular serotypes associated with invasive pneu-
47F, 47A, 48
mococcal disease and current vaccines only cover up to 13 of these Streptococcus agalactiae Ia, Ib, II, III, IV, V, VI, VII, VIII, IX
(Table 2). Furthermore, although there are vaccines under
 K. Diallo et al. / EBioMedicine 65 (2021) 103274 3

Fig. 1. Approaches used in the diagnosis of meningitis from cerebrospinal fluid.

Figure created with BioRender.com.

of invasive disease due to Hib [8]; (ii) The decrease in serogroup C relevant measures are implemented for case and outbreak manage-
meningococcal disease in countries that implemented the serogroup ment and also informs future vaccine development. A global initiative
C conjugate vaccine; (iii) The near disappearance of serogroup A aimed at eliminating meningitis worldwide by 2030 has recently
meningococcal infections in the African meningitis belt; and (iv) been endorsed by the WHO (https://www.who.int/activities/defeat
Major reductions in invasive pneumococcal disease globally ing-meningitis-by-2030). One of five pillars of the roadmap for this
[4,9 11]. global vision is “Diagnosis and treatment” and it highlights the need
Given the diversity of organisms that can cause meningitis, accu- for comprehensive, cost-effective diagnostic approaches to enhance
rate species and capsule identification is essential for diagnosis, treat- surveillance [12]. A number of diagnostic assays have been developed
ment, surveillance, and public health intervention. This ensures that (Fig. 1). Here, we provide an account of recently published molecular
4 K. Diallo et al. / EBioMedicine 65 (2021) 103274

diagnostic assays and report on the developments made in this field. The first molecular methods to be developed were PCR assays that
Clinicians and diagnostic laboratories can use this information to detect a single pathogen, but these have been largely superseded by
make decisions on assay suitability and identify areas that require multiplex PCR assays (simultaneous testing of multiple targets) such
further research. as rtPCR assays, which reduces time and consumable costs [23]. Note
that the tests described below are not commercially available except
1.1. Point-of-care assays where indicated.

Characterisation of organisms by culture from a normally sterile 1.2.1. Haemophilus influenzae

site, such as cerebrospinal fluid (CSF), has long been the mainstay of Several singleplex PCR assays detecting Hib have been described,
the laboratory confirmation of suspected bacterial meningitis [2]. including those that detect: the capsule gene bexA [24]; the outer
However, several factors impede the use of CSF culture for diagnosis, membrane proteins OmpP2 (ompP2) and OmpP6 (ompP6) [25,26];
including: the fact that it can take 24 48 h or longer to obtain culture the phase variable gene licA encoding phosphorylcholine kinase [25];
results [13]; suboptimal CSF specimen volume; fastidious, difficult to the protein D gene (hpd) [26,27]; and fuculokinase (fucK) [25]
cultivate organisms; suboptimal storage and transportation; and (Table 3). Although assays targeting fucK and hpd have been shown
prior antimicrobial administration resulting in non-viable bacteria. to reliably detect Hib [25,28], some non-typeable H. influenzae (NTHi)
Consequently, a variety of approaches have been developed for the strains have been reported to lack either fucK [29] or hpd genes [30]
diagnosis of bacterial meningitis, including: latex agglutination, lat- while some Haemophilus parainfluenzae strains were reported to pos-
eral flow assays, and molecular diagnostic assays (Fig. 1). sess hpd [28] (Table 3). The ompP2 and ompP6 sequences also display
Although sero-agglutination assays are among the simplest tests some diversity, making them difficult targets to detect reliably, and
to execute, their sensitivity can be reduced if the patient received there is some evidence for cross-reactivity with Haemophilus haemo-
antimicrobial treatment prior to specimen collection [14]. Agglutina- lyticus [31]. The increasing availability of whole genome sequence
tion assays also depend on expression of the target antigen, most data (WGS) will provide further opportunities to determine the prev-
often the polysaccharide capsule which bacteria can modulate both alence, diversity and distribution of these genes within the genus
in vitro and in vivo [15,16]. Cross-reactivity due to poly-agglutination Haemophilus.
can be problematic for some sero-agglutination assays and can pre-
vent definitive characterisation of the isolate [17]. Lateral flow assays 1.2.2. Neisseria meningitidis
(LFAs; one example is the commercial pregnancy test), consist of pre- Recommended genetic targets for the identification of meningo-
fabricated strips containing dry reagents that are activated by appli- cocci include the superoxide dismutase gene sodC and the capsule
cation of a fluid sample [18 20]. Several types of LFAs exist and the gene ctrA (Table 3) [32,33]. Although sodC is ubiquitous in meningo-
greatest benefits of these tests are that they are rapid, single use, and cocci, it has been reported to lead to false positive results due to the
point-of-care. The different types of LFAs include those in which anti- presence of sodC homologues in other bacterial species including H.
bodies are used as recognition elements (‘lateral flow immunoas- influenzae [33], while the ctrA gene preferentially detects encapsulated
says’). An example of this is the MeningoSpeed rapid diagnostic test, meningococci (Table 3). As a result, other tests have been developed,
an immunochromatographic test that can detect meningococcal using the outer membrane protein gene porA and the capsule null
serogroups from CSF samples [21]. Other LFAs consist of antigen-anti- locus (cnl), the combination of which identifies unencapsulated
body interactions with specific tagged doubled-stranded amplicon meningococci, which cause invasive disease rarely, but which are com-
detection after PCR (‘nucleic acid lateral flow immunoassay’) or monly found in asymptomatic carriage [33,34]. In such situations, the
assays where specific nucleic acid amplicons are hybridised with host may have a predisposing immunosuppressive condition [35];
immobilised complementary probes (‘nucleic acid lateral flow assay’) however, this also suggests that rtPCR assays that only test for encap-
[20]. Although the sensitivity of sero-agglutination and LFAs is sus- sulated meningococci may be overlooking cnl strains, and that a com-
ceptible to bacterial viability, the ease and rapidity with which these bined cnl and ctrA assay may be needed to increase sensitivity.
assays can be used make these assays desirable in more challenging
low- and middle-income countries (LMIC). 1.2.3. Streptococcus pneumoniae
The genetic targets used to detect S. pneumoniae include the auto-
1.2. Endpoint PCR and real-time PCR assays for the detection of lysin gene lytA and the permease gene of the Pia ABC transporter,
meningitis pathogens piaB, although the latter was reported to be absent in some nontype-
able or serotype 6B pneumococci [36]. Consequently, lytA was the
Molecular tools including the polymerase chain reaction (PCR), recommended genetic target for pneumococci at the time of writing
real-time PCR (rtPCR), qualitative or quantitative PCR (qPCR), and [36]. It has been reported that lytA homologues can be present in
loop-mediated isothermal amplification assays (LAMP) have the closely-related species of Streptococcus [37], therefore an assay tar-
potential to overcome many of the limitations of culture-based geting a putative transcriptional regulator of the GntR-family (gene
approaches, as they target bacterial DNA and are not constrained by SP2020), belonging to the core genome of the pneumococcus, was
the presence of cultivable organisms. These molecular tools are now developed [36] (Table 3). Finally, the pneumolysin gene, ply, as well
the methods of choice for many laboratories and have improved pub- as the pneumococcal surface adhesion gene, psaA, have been
lic health measures because they involve standard, generic laboratory described as targets for the detection of pneumococci in rtPCR assays;
techniques that allow multiple pathogens to be rapidly detected. however, strains of Streptococcus pseudopneumoniae and viridans
Indeed, the World Health Organisation (WHO) recommends the use group streptococci can also be positive for these genes, precluding
of rtPCR in the testing of pneumococcus, meningococcus, and Hib their use as reliable genetic targets [38].
from suspected cases of meningitis [13]. Major challenges remain,
however, in the deployment of these assays in LMICs, due to the vari- 1.2.4. Streptococcus agalactiae
ability of laboratory capabilities, shortage of trained laboratory per- Singleplex assays detecting GBS have also been developed,
sonnel and challenges in procurement of reagents and equipment. although many of these are optimised for identifying the bacterium
Progress has been made through initiatives such as MenAfriNet from gut or vaginal colonisation and/or shortly after labour rather than
(www.menafrinet.org) and laboratories in several countries in the for the diagnosis of acute infection [39,40]. Four commercial assays are
African meningitis belt and beyond have acquired the capability to available: (i) The Becton Dickinson MAX GBS assay; (ii) The ARIES GBS
perform these assays [22]. assay from Luminex Corporation; (iii) The Illumigene Group B
 K. Diallo et al. / EBioMedicine 65 (2021) 103274 5

Table 3
Genetic targets used in the detection of the four main causes of bacterial meningitis.

Bacterial species Genetic target and type of Function Disadvantage (if known) Sensitivity (%) Specificity (%) References
assay

Haemophilus influenzae (Hib) rtPCR

bexA ATP-binding protein (cap- Does not detect NTHi strains 100 90 100 [21]
sule region I)
omP2 Outer membrane protein P2 Less sensitive than fucK or 97.1 100 [25]
licA, and requires very
high genome copies for
detection
omP6 Outer membrane protein P6 Nucleotide sequence NA NA [31]
diversity
licA Protein LicA Fails to detect some serotype 97.1 99.1 [25]
e isolates
hpd Protein D May be absent in some NTHi 95/95.7 91/ 92.3 [30]
strains
fucK Fuculokinase Deletion of the fucose 97.1 100 [29]
operon in some strains
and reactivity with H.
aegyptius
LAMP:
Hib capsule 247 bp region of the capsule 100 100 [62]
pstA Phosphate transport system 80 100 [68]
permease protein
Neisseria meningitidis rtPCR:
(meningococcus) ctrA Capsule transport (region I Only encapsulated meningo- 71.6 NA [33]
capsule locus) cocci have ctrA
sodC Superoxide dismutase Less sensitive in sterile body 99.6/ 94.7 100/ 77.9 [32]
fluids, for use in conjunc-
tion with ctrA to improve
sensitivity
Some species possess
homologous sodC genes
crgA Transcriptional regulator of Can be found in N. 93 96 [87]
LysR family gonorrhoeae
porA Outer membrane porin, PorA Can be found in N. gonor- 96.1 91.6 [88,89]
rhoeae and may be absent
in some meningococci
LAMP:
ctrA Capsule transport 89/100 100/98.9 [64,65]
NMO_1242 Putative cytolysin secretion 100 100 [68]
ABC transporter protein
Streptococcus pneumoniae rtPCR:
(pneumococcus) lytA Autolysin Possible homologues in 100 99.5/100 [36,38]
other closely-related spe-
cies leading to false
positives
piaB Pia ABC transporter piaB is absent in some pneu- 95.3 99.5 [36]
mococci including some
serotype 6B strains and
absent in some non-type-
able strains
GntR-family SP2020 Putative transcriptional reg- May be present in non- 100 99.8 [36]
ulator gene pneumococcal strains;
combining with lytA
results increases
sensitivity
ply Pneumolysin Can lead to false-positive 100 81 [38]
reactions in the presence
of viridans group
streptococci
LAMP:
lytA Autolysin 100 100 [66]
SPNA45_01710 Heparinase II/III-like protein 95.7 100 [68]
Streptococcus agalactiae rtPCR:
(GBS) cfb S. agalactiae CAMP factor some CAMP-negative GBS NA NA [41]
may not carry the cfb gene
dltS Histidine kinase specific to 96.1 95.9 [59]
GBS
LAMP:
cfb S. agalactiae CAMP factor NA NA [41]
6 K. Diallo et al. / EBioMedicine 65 (2021) 103274

Streptococcus assay from Meridian Bioscience; and (iv) The Xpert GBS capsule-specific disease burden and guiding vaccination decisions.
LB assay produced by Cepheid Inc. Three of these use rtPCR assays, Given that the majority of invasive disease cases caused by H. influen-
while Illumigene uses a loop-mediated isothermal amplification zae are due to serotype b, few assays detecting the remaining five
(LAMP) assay with the cfb gene as the primary target. The gene cfb enc- capsules have been developed, although a PCR-endpoint based assay
odes the extracellular pore-forming toxin also known as the CAMP fac- can be used to detect H. influenzae serotypes [52,53]. Multiple capsu-
tor [40]. The Christie-Atkinson-Munch-Peterson (CAMP) test has been lar polysaccharides are associated with invasive disease and, differen-
the conventional culture-based test for identifying GBS and differenti- tiation of these capsules is essential (Table 2). Assays have been
ates haemolytic versus non-haemolytic GBS. Notably, some GBS iso- developed that detect meningococcal or pneumococcal capsules
lates are CAMP negative and lack cfb, indicating that further studies associated with invasive disease [45,54]. Whilst rtPCR assays to
assessing the distribution of cfb are required [41,42]. Another assay detect meningococcal serogroups A, B, C, W, Y, and X have been devel-
employed a recombinase polymerase amplification (RPA) method to oped, the complexity of the pneumococcal capsular locus and large
detect the cfb gene in vaginal swabs and this assay was sensitive and number of serotypes make the design of multiple PCR assays capable
specific for GBS [43]. All these assays required samples to be enriched of detecting a large number of potential serotypes a major challenge
in Lim Broth for 18 24 h prior to testing and results demonstrated [55,56]. Most assays focus on detecting a subset of the prevalent sero-
greater sensitivity compared with culture alone. types circulating in a region and/or those most frequently associated
with invasive disease. An alternative approach determines serotypes
1.3. Multiplex assays based on the capsule polymerase gene wzh [57].
There are ten GBS serotypes and six serotypes (Ia, Ib, II, III, IV and
An advantage of using PCR assays in infectious disease diagnostics V), in particular, are associated with invasive disease. Few rtPCR
is that multiple pathogens may be targeted in a single assay, which assays have been designed to detect the presence of S. agalactiae
conserves clinical specimens, saves time, and reduces costs. Multiplex with one based on the dltS gene, a sensor protein, and capsule-spe-
rtPCR assays that detect Hib, meningococci and pneumococci in one cific genes encoding serotypes Ia, Ib, and III [58,59]. The assay is spe-
reaction have been developed and can be used to test CSF directly and cies-specific for GBS with no cross-reaction with other closely related
avoid the need for DNA extraction [44,45]. One prototype assay has Streptococcus spp and it reliably detects serotypes Ia, Ib, and III.
been developed to detect six microorganisms associated with menin-
gitis including (molecular target): S. pneumoniae (lytA); N. meningitidis 1.5. Loop-mediated isothermal amplification assays (LAMP)
(ctrA); H. influenzae (ompP2); S. agalactiae (cfb); Listeria monocytogenes
(iap); and Cryptococcus neoformans (18S rDNA) [46]. To date, the assay The loop-mediated isothermal amplification (LAMP) assay was
has been tested using a limited number of specimens and therefore developed in 2000 [60]. This assay amplifies a specific DNA target
requires further validation; however, of the 45 suspected cases of bac- and has the advantage of working in isothermal conditions, removing
terial meningitis, the causative agent was identified in 32 CSF speci- the requirement for thermocyclers. LAMP uses a DNA polymerase
mens using a combination of phenotypic and genotypic tests and of that has strand displacement activity, allowing it to separate the dou-
these 16 were identified solely on the basis of molecular assays [46]. ble-stranded DNA without the need for a temperature change and
More recently, a commercial TaqMan Array card has been devel- thus the reaction can be conducted in a simple water bath. In addition
oped that can detect multiple meningitis-associated pathogens [47]. to requiring very basic equipment, LAMP assays have been found to
This assay was tested in CSF samples originating from West Africa be highly specific [61] and their efficiency is less affected by back-
and results indicated that diverse infectious aetiological agents were ground DNA [60]. As a result, several LAMP assays have been
present. Another commercially available test is the BioFireÒ designed for the diagnosis of bacterial pathogens, including the four
FilmArrayÒ Meningitis/Encephalitis (ME) Panel which can detect 14 pathogens included in this review.
pathogens (seven viruses, six bacteria and one fungus) simulta- A LAMP assay to detect Hib directly from CSF was described in
neously from CSF samples, including the four bacteria that are the 2011 [62], which targeted a 247 bp nucleotide sequence of the cap-
focus of this review [48]. Performance of the ME Panel was evaluated sule locus and was performed at 65 °C for 35 min. The method was
using clinical CSF samples that had previously tested positive using evaluated using a collection of H. influenzae isolates, other Haemophi-
routine methods: the ME Panel resulted in an overall positive agree- lus species, and other genera, as well as stored CSF samples originat-
ment of 97.5% for bacterial pathogens [48]. A study in Ethiopia involv- ing from suspected meningitis cases. The Hib LAMP assay
ing 218 patients with suspected meningitis identified S. pneumoniae, discriminated Hib from other encapsulated H. influenzae strains and
N. meningitidis and H. influenzae using the ME Panel, but only in 5 was more sensitive than the bexA PCR assay and a nested PCR for the
(2.2%) cases [49]. Although the ME Panel has been shown to increase detection of Hib [62]. Another study used a similar design to develop
pathogen detection rate, it has been noted that interpretation and five LAMP assays for the characterisation of non-serotype b H. influ-
correlation of results requires experienced users and it does not allow enzae isolates [63]. Validation was performed using a collection of H.
the capsular polysaccharide to be determined [50]. Furthermore, the influenzae isolates, other Haemophilus species, other genera, and
ME Panel may be cost-prohibitive for LMICs since the estimated aver- spiked CSF samples. This non-serotype b LAMP assay was as specific
age cost is currently around $239 per test (around £180/€200) [51]. and sensitive as the comparable non-serotype b PCR assay, and
It should also be noted that there are several other aetiological results were confirmed by dideoxy nucleotide sequencing of the
agents of meningitis, some of which are highly prevalent in LMIC set- products [63].
tings, including Cryptococcus neoformans; Mycobacterium tuberculo- A LAMP assay for the detection of N. meningitidis uses primers that
sis; Salmonella enterica var Typhi; Herpes simplex virus; Varicella target the ctrA gene. This assay performed as well as the standard
zoster virus and enteroviruses. C. neoformans and M. tuberculosis in rtPCR, but at a fraction of the cost and time [64]. The method was
particular are common among HIV-infected individuals. Inexpensive, assessed as a point-of-care diagnostic tool at the emergency depart-
easy to use multiplex assays with the capacity to detect all of these ment of the Royal Belfast Hospital for Sick Children, UK [65]: A total
pathogens need to be developed. of 161 patients had nasopharyngeal and blood samples tested using
the LAMP assay in addition to the routine culture and PCR but the
1.4. PCR and rtPCR assays for the detection of capsular types assay was not validated for CSF specimens. Similarly, a LAMP assay
targeting the lytA gene of S. pneumoniae was developed and validated
Once the bacterial species has been confirmed, further PCR assays using a set of reference strains that included different Streptococcus
are available to detect capsule types, which is important in assessing species and other genera, clinical alpha-haemolytic streptococcal
 K. Diallo et al. / EBioMedicine 65 (2021) 103274 7

isolates, and CSF samples from suspected meningitis cases [66]. The 1.6. Sensitivities and specificities of published assays
lytA LAMP assay was found to be more sensitive than the comparable
lytA PCR. The sensitivity and specificity values of various singleplex rtPCR
Multiplex LAMP assays have been more difficult to design assays were high, ranging from 91 to 100% (Table 3). Multiplex assays
because of the non-exonuclease activity of the polymerase enzyme, targeting S. pneumoniae, N. meningitidis and H. influenzae were
which prevents the use of labelled probes; however, the ability to reported to have sensitivities ranging from 73 to 94%, and good spe-
detect multiple pathogens in a single reaction has been explored in cificities (98 to 100%), comparable to those of singleplex rtPCR assays.
the context of meningitis. A prototype LAMP assay was designed to LAMP assays were found to generate both higher sensitivity and
detect S. pneumoniae, Staphylococcus aureus, Streptococcus suis, and specificity values compared to rtPCR (Table 3). The accuracy of LAMP
GBS based on the sequence diversity of the 16 rRNA genes of each assays was consistently high (sensitivity of 80 100% and specificity
bacterial species [67], which exhibited better sensitivity and a lower of 99 100%) using various clinical samples including CSF, blood and
limit of detection than conventional PCR. It was specific, although nasopharyngeal swabs from children.
only three other bacterial species were tested for cross-reactivity Among PCR-based tests, the sensitivity and specificity values were
(N. meningitidis, H. influenzae, and Escherichia coli). The assay was higher for CSF samples than for either blood samples or oro-nasopha-
validated with DNA extracted from cultured isolates but was not ryngeal swabs for the diagnosis of meningitis; however, using LAMP
tested on CSF. assays that target ctrA loci for the diagnosis of invasive meningococ-
A modified LAMP assay, the Tth Endonuclease Cleavage (TEC) cal disease, the sensitivity and specificity values were similar regard-
LAMP, has been developed, allowing simultaneous detection of N. less of the sample used [65]. Nonetheless, further studies are needed
meningitidis, S. pneumoniae, and H. influenzae [68]. This assay includes to clearly elucidate whether other non-CSF specimens are suitable
a thermostable enzyme capable of cleaving abasic sites in double- for LAMP diagnosis of meningococcal disease.
stranded DNA, along with a TEC primer/probe, which acts as a LAMP
forward inner primer, but which also contains an abasic site, a fluo- 2. Outstanding questions
rescent dye, and a quencher [68]. The addition of the fluorescent dye
allows multiplexing and real-time monitoring of the amplification. In the longer term, whole genome sequencing (WGS) will become
The assay targets the genes encoding: (i) A heparinase II/III-like pro- more routinely employed in the diagnosis of infectious diseases,
tein in S. pneumoniae (SPNA45_01710); (ii) A putative cytolysin secre- including meningitis [74]. Ultimately, this will likely include the
tion ABC transporter protein in N. meningitidis (NMO_1242; hylB); development of non-culture-based methods that directly sequence
and (iii) A phosphate transport system permease protein in Hib pathogen genomes from clinical specimens. Current requirements for
(pstA). A random gene fragment is incorporated as an internal control these approaches include DNA extraction protocols that deplete
[68]. This prototype assay was validated using a panel of DNA human DNA and increase the bacterial sequence yield [75]. Such
extracted from reference and clinical isolates and tested in PCR-con- assays will have the benefit of allowing multiple genetic targets to be
firmed cases of S. pneumoniae, N. meningitidis, and H. influenzae infec- analysed directly from genomes, enabling the simultaneous identifi-
tions. The specificity was 100% for each target and pathogen-specific cation of the pathogen, the predicted capsular type, antimicrobial
sensitivity was 95.7% for S. pneumoniae, 100% for N. meningitidis and resistance determinants, etc. Progress has been made in this respect
80% for H. influenzae. Although the reactions were conducted in a with selective whole-genome amplification (SWGA), an isothermal
rtPCR thermocycler, this assay could also be performed with simpler multiple-displacement amplification-based method, validated in 12
point-of-care technologies that allow sample heating and fluorescent CSF specimens from invasive meningococcal disease cases [76]. In
detection [69]. An improvement of the (TEC)-LAMP method is loop- addition, metagenomic next generation sequencing is also showing
primer endonuclease cleavage (LEC)-LAMP, which allows single promise in improving the diagnosis of meningitis and encephalitis
nucleotide polymorphisms to be detected in either singleplex or mul- [77,78]. Oxford Nanopore sequencing technology was recently
tiplex assays [70]. This assay successfully resulted in the simulta- employed in Zambia for the rapid diagnosis of bacterial meningitis
neous detection of N. meningitidis, S. pneumoniae, and H. influenzae species through the sequencing of 16S rRNA [79].
and may be useful in the detection of allele-specific differences To reach the goal of implementing WGS in LMICs, a range of tech-
between and within bacterial species in a single assay. Finally, a com- nical, financial, and infrastructural challenges will have to be met
mercial LAMP assay called the eazyplexÒ CSF direct panel is available and, until then, it is unlikely laboratories in LMICs will be able to rou-
and has the capacity to detect several pathogens associated with tinely undertake WGS for the diagnosis of meningitis. In the immedi-
meningitis, including E. coli, H. influenzae, L. monocytogenes, N. menin- ate future, the most promising opportunities involve refining
gitidis, S. agalactiae, and S. pneumoniae. The average cost of this assay molecular diagnostic assays using comparative genomic analyses to:
was estimated to be around $62 (around £45/€50) per sample and (i) Improve practices of sampling, storage and transport of speci-
was relatively easy to use with a sensitivity of 90.9% and specificity of mens; (ii) Improve the performance of existing targets by increasing
100% [71]. sensitivity and specificity; and (iii) Identify alternative diagnostic tar-
Ultimately, LAMP technology could be developed into affordable gets to enhance species-specificity [80]. In order to undertake such
point-of-care devices such as paper-based LAMP assays that allow activities, an enhanced representation of genomes originating from
the simultaneous detection of S. agalactiae, S. pneumoniae and S. LMICs needs to be established and initiatives such as the Global Men-
aureus using dry reagents that are easy to store and transport in ingitis Partnership seek to address this issue [81]. Furthermore, the
LMICs [72]. Improvements in LAMP methodologies that could allow analysis and comparison of genomes relies on the availability of data-
for real-time monitoring system without post-amplification manipu- bases where genomes can be stored, curated and analysed. For exam-
lations make these assays potential candidates for the molecular ple, PubMLST databases (https://pubmlst.org) store and annotate
diagnosis of infectious diseases such as meningitis. The pooled high thousands of genomes (with associated metadata) of all four bacterial
sensitivity and specificity values determined from a review of LAMP species included in this review in dedicated genome libraries [82]
assays detecting the meningococcus are encouraging [73] and the and BMGAP allows users to deposit and access N. meningitidis and H.
design of a multiplex LAMP assay including all four pathogens influenzae genomes using a secure online portal [83].
included in this review is now a possibility. Such an assay would be The timely diagnosis of the aetiological agent in meningeal infec-
invaluable for use in hospital settings and the lack of requirements tions is essential to facilitate treatment, patient management, and
for expensive equipment makes it an appealing approach for labora- improved clinical outcomes. The most desirable diagnostic assay
tories with limited resources. would be one in which multiple pathogens can be detected in a cost-
8 K. Diallo et al. / EBioMedicine 65 (2021) 103274

effective, easy to use system that provides rapid and robust results. [5] Bruce MG, Deeks SL, Zulz T, Navarro C, Palacios C, Case C, et al. Epidemiology of
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CMCR, OR, MKT, XW: contributed to writing strip technology for detection of infectious agents and chemical contaminants: a
KD, OBH, VFF contributed to figures, data analysis and interpreta- review. Anal Bioanal Chem 2010;397(3):1113–35.
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Declaration of Competing Interest [21] Haddar CH, Terrade A, Verhoeven P, Njanpop-Lafourcade BM, Dosso M, Sidikou F,
et al. Validation of a new rapid detection test for detection of Neisseria meningiti-
dis A/C/W/X/Y antigens in cerebrospinal fluid. J Clin Microbiol 2020;58(3).
The authors confirm there are no conflicts of interest. [22] Feagins AR VJ, Fernandez K, Njanpop-Lafourcade BM, Mwenda JM, Osee Sanogo Y,
Paye MF, Payamps SK, Mayer L, Wang X. The Strengthening of laboratory systems
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Acknowledgments perspective. J Infect Dis 2019;220:S175–S81.
[23] Wang X, Theodore MJ, Mair R, Trujillo-Lopez E, du Plessis M, Wolter N, et al. Clini-
cal Validation of multiplex real-time PCR assays for detection of bacterial menin-
OBH, MJCM, ABB, VF, LI and KD were funded by the Department of gitis pathogens. J Clin Microbiol 2012;50(3):702–8.
Health and Social Care using UK Aid funding and is managed by the [24] Wroblewski D, Halse TA, Hayes J, Kohlerschmidt D, Musser KA. Utilization of a
NIHR (NIHR PR-OD-1017 20007). The views expressed in this publi- real-time PCR approach for Haemophilus influenzae serotype determination as an
alternative to the slide agglutination test. Mol Cell Probes 2013;27(2):86–9.
cation are those of the authors and not necessarily those of the
[25] Meyler KL, Meehan M, Bennett D, Cunney R, Cafferkey M. Development of a diag-
Department of Health and Social Care. KD is supported by the DELTAS nostic real-time polymerase chain reaction assay for the detection of invasive
Africa Initiative [Afrique One-ASPIRE/DEL-15 008]. The findings and Haemophilus influenzae in clinical samples. Diagn Microbiol Infect Dis 2012;74
(4):356–62.
conclusions in this report are those of the authors and do not neces-
[26] Wang X, Mair R, Hatcher C, Theodore MJ, Edmond K, Wu HM, et al. Detection of
sarily represent the official position of the Centers for Disease Control bacterial pathogens in Mongolia meningitis surveillance with a new real-time
and Prevention. PCR assay to detect Haemophilus influenzae. Int J Med Microbiol 2011;301
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