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SUPPLEMENT ARTICLEThe Journal of Infectious Diseases S190 jid 2019:220 (Suppl 4) • Sanogo et al Correspondence: X. Wang, PhD, Chief, Bacterial Meningitis Laboratory, Meningitis and Vaccine Preventable Disease Branch, Division of Bacterial Diseases, Natio nal Center for Immunization and Respiratory Diseases, Center for Disease Control and Pr evention, 1600 Clifton Road NE, MS D-11, Atlanta, GA 30333 (gqe8@cdc.gov). a Y. O. S. and I. G. contributed equally to this work. Presented in part: 2018 International Conference of Emerging Infectious

Diseases, August

26-29, 2018, Atlanta, GA.

The Journal of Infectious Diseases

2019;220(S4):S190-7

Published by Oxford University Press for the Infectious Diseases Society of America 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US. This Open Access article contains public sector information licensed und er the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/2/).

DOI: 10.1093/infdis/jiz272

A New Sequence Type of

Neisseria meningitidis

Serogroup

C Associated With a 2016 Meningitis Outbreak in Mali

Yibayiri Osee Sanogo,

1 a

Ibréhima Guindo,

2 a

Seydou Diarra,

2

Adam C. Retchless,

1

Mahamadou Abdou,

2

Souleymane Coulibaly,

2

Mahamadou Farka Maiga,

3

Mama Coumaré,

3

Bakary Diarra,

4

Alexander Chen,

1

How-Yi Chang,

1

Jeni T. Vuong,

1

Anna M. Acosta,

1

Samba Sow,

4 5

Ryan T. Novak,

1 and Xin Wang 1 1 Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, Nati onal Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention,

Atlanta, Georgia;

2 Institut National de Recherche en Santé Publique, Bamako, Mali; 3

Direction Nationale de la Santé, Bamako, Mali;

4

Ministère de la Santé, Mali;

5

Centre National d'Appui et de

Lutte contre les Maladies/Centre des Vaccins en Dévelopement, Mali

In 2016, Mali reported a bacterial meningitis outbreak consisting of 39 suspected cases between epidemiologic weeks 9 and 17

with 15% case fatality ratio in the health district of Ouéléssebougou, 80 kilometers from the capital Bamako. Cerebrospinal ?uid

specimens from 29 cases were tested by culture and real-time polymerase chain reaction; 22 (76%) were positive for bacterial men

ingitis pathogens, 16 (73%) of which were

Neisseria meningitidis

(Nm). Of the Nm-positive specimens, 14 (88%) were

N meningitidis

serogroup C (NmC), 1 was NmW, and 1 was nongroupable. Eight NmC isolates recovered by culture from the outbreak were

characterized using whole genome sequencing. Genomics analysis revealed that all 8 isolates belonged to a new sequence type (ST)

12446 of clonal complex 10217 that formed a distinct clade genetically similar to ST-10217, a NmC strain that recently caused large

epidemics of meningitis in Niger and Nigeria. ?e emergence of a new ST of NmC associated with an outbreak in the African men

ingitis belt further highlights the need for continued molecular surveillance in the region. Keywords. African meningitis belt; Neisseria meningitidis; outbreak; serogroup C; sequence type.

Neisseria meningitidis

is a Gram-negative diplococcal bacterium that can cause life-threatening invasive meningococcal disease (IMD). Invasive meningococcal disease represents a serious public health threat worldwide, and it can present as meningitis, bacteremia, or bacteremic pneumonia. The highest burden of meningococcal meningitis is in the African meningitis belt, a region spanning from Senegal to Ethiopia that experiences seasonal meningitis outbreaks punctuated by periodic epidemics every 5-12 years [

1, 2].

Neisseria meningitidis

is classi?ed into 12 serogroups based on the structure of its polysaccharide capsule [ 3 ]. Six serogroups (A, B, C, W, Y, and X) cause the vast majority of IMD cases. Before the introduction of the meningococcal serogroup A con jugate vaccine ([MACV] MenAfriVac) in 2010, most menin gitis cases in the African meningitis belt were caused by N meningitidis serogroup A (NmA). Mass vaccination by MACV resulted in a signi?cant reduction of NmA cases and elimina tion of NmA epidemics in the region [ 4

]. Despite this success, non-NmA serogroups, including NmW, NmX, and NmC, re-main a threat in the region and continue to cause periodic

outbreaks and epidemics [ 5-9 ]. It is worth noting that NmC has emerged as an increasing risk for countries of the African continent both within [ 10 ] and outside the belt [ 11 Historically, cases of meningitis caused by NmC have been in frequently reported in the African meningitis belt. ?e ?rst ep idemic associated with NmC was reported in 1975 in northern Nigeria, where 53% of all suspected cases were attributed to NmC [ 12 ]. Localized outbreaks of meningitis caused by NmC have been reported in Burkina Faso in 1979 [ 13 ], in Mali be tween 1988 and 1992 [ 14 ], and in Nigeria in 2013-2014 [ 15 , 16].

Neisseria meningitidis

serogroup C emerged as a major public health threat when it caused a large epidemic in Niger in 2015 with 9367 suspected cases and 549 deaths [ 17 ]. ?e largest men ingitis epidemic primarily caused by NmC (83%) was reported in the northern region of Nigeria in 2017 with 14 518 reported suspected cases and 1166 deaths [ 18 ]. Molecular typing re vealed that these recent epidemics were caused by a new NmC strain, sequence type (ST)-10217 belonging to clonal complex (CC) 10217 [ 17 , 19]. Comparative genomics analysis indicated that NmC ST-10217 emerged from an asymptomatically and nasopharyngeally carried meningococcal strain, through the ac quisition of virulence genes such as the NmC capsule genes and a meningococcal disease-associated prophage (MDAΦ) [ 20 Mali, a country in the northwestern part of the meningitis belt, has experienced 17 major epidemics of meningococcal meningitis since 1940, when the disease was first reported in applyparastyle "g//caption/p[1]" parastyle "FigCapt"

220Downloaded from https://academic.oup.com/jid/article/220/Supplement_4/S190/5610768 by guest on 25 October 2023

Outbreak of Neisseria meningitidis Serogroup C ST-12446 in Mali • jid 2019:220 (Suppl 4) • S191

the country [

1, 21]. The largest epidemics in Mali occurred

between 1969 and 1971, with 18 288 suspected cases and

1191 deaths [

22
], and in 1997, with 11 228 suspected cases and 1126 deaths [ 21
, 23]. The last outbreak reported in Mali occured in 2008 in Ouéléssebougou, a health district located approximately 80 kilometers south-east of the cap ital Bamako. Fifty-one suspected cases were reported, with a case fatality ratio (CFR) of 12.7%, and the outbreak was due primarily to NmA. However in 2015, 7 NmC cases were reported from the region of Gao, and in 2016, Mali reported a localized outbreak of meningococcal meningitis prima rily due to NmC in the health district of Ouéléssebougou. We describe the 2016 outbreak and characterize the avail able isolates using whole genome sequencing and molecular typing. These isolates were also compared with other strains circulating in the African region.

MATERIALS AND METHODS

The Health District of Ouéléssebougou

The health district of Ouéléssebougou (

Figure 1

) covers an area of approximately 1118 square kilometers with a population of

215 775 inhabitants. Ouéléssebougou is one of the 10 initial

health districts in Mali supported by the MenAfriNet project, an international consortium funded by the Bill & Melinda Gates Foundation with the purpose of reinforcing case-based men

ingitis surveillance systems in 5 key countries in the African meningitis belt and providing a platform to monitor the impact of MACV [24].

Data and Specimen Collection

Epidemiological data, including patient's residence, age, and gender as well as date of disease onset, symptoms, and vac cination status for each suspected meningitis case was col lected on standardized case report forms at district levels. Cerebrospinal fluid (CSF) specimens were collected for each reported suspected case [ 25
]. Ethics approval and participant consent was not necessary because specimens were collected for purposes of disease surveillance by Mali Ministry of Health and were deidentified before shipping to the Centers for Disease Control and Prevention ([CDC] Atlanta, GA). As part of the outbreak response, the first 6 CSF specimens collected during the investigation were transported to Bamako and analyzed by Pastorex and cytology at the Center for Vaccine Development laboratory of the National Hospital at Gabriel Touré and subsequently confirmed by culture and real-time polymerase chain reaction (rt-PCR) at the National Reference Laboratory, Institut National de Recherche en Santé Publique (INRSP) in Bamako. The other specimens were analyzed at INRSP using Gram staining and cytology and confirmed by culture and rt-PCR. Whole genome sequencing and Sanger sequencing were performed at the World Health Organization (WHO) Collaborating Center at the CDC in Atlanta. To 25
20

16 cases

4 cases

1 case

Mauritania

SenegalCity of

Gao

City of

AnsongoGaoRegion

Burkina Faso

Bougouni District

Ouéléssebougou

DistrictCity of

Bamako

GuineaNiger

City of

FanaAlgeria

Type ND ST -10217

ST-12446

15 10 -15-10-5 Long Lat 05

Figure 1.

Distribution of Neisseria meningitidis serogroup C strains in Mali during 2015-2017. Sequence type (ST)-1

0217 (in green) was detected in the Gao region, and

ST-12446 was detected in the Ouéléssebougou district. The STs that were not determined ([ND] in red) were detected in the Gao regi

on.Downloaded from https://academic.oup.com/jid/article/220/Supplement_4/S190/5610768 by guest on 25 October 2023

S192 • jid 2019:220 (Suppl 4) • Sanogo et al identify additional NmC cases in Mali, we reviewed and analyzed national laboratory data from 2015 to 2017. Molecular Characterization by Whole Genome Sequencing and Sanger

Sequencing

For whole genome sequencing, deoxyribonucleic acid (DNA) was extracted from isolates using QIAGEN Gentra Puregene kit (QIAGEN, Hilden, Germany) and sheared to 600 base pairs (bp) using Covaris ultrasonicator (Covaris Inc., Woburn, MA). Dra? genome sequences were generated from 250-bp, paired- end read data generated by an Illumina HiSeq 2500 (CDC Biotechnology Core Facility) as previously described [ 26
Genome assemblies of the 8 isolates from Ouéléssebougou were published on PubMLST [ 27
] with identi?ers

58393-58397,

58399, and 58400.

Genes encoding outer membrane proteins and vaccine antigens and multilocus sequence typing (MLST) genes were sequenced and identi?ed using the PubMLST Genome

Comparator tool [

27
]. Porin A (PorA), Porin B (PorB), and Ferric enterobactin transport (FetA) types are de?ned by their respective variable regions [ 28
]. Molecular pro?le of an iso late was de?ned as serogroup: PorA:FetA:ST(CC) in this study. Neisserial adhesin A (NadA), neisserial heparin binding antigen (NhbA), and factor H binding protein (FHbp) were identi?ed as described in PubMLST [ 29

Phylogenetic Analysis

To understand how the Mali outbreak strains are related to other strains in Mali and in the African continent, a phylogeny was constructed using sequences of the 8 Ouéléssebougou isolates along with sequences of 1 NmC isolate from Gao, 76 isolates from Niger 2015 [ 19 ], 73 isolates from Nigeria 2013-

2017 [

20 ], and 1 ST-9367 carriage isolate from Burkina Faso

2012 (BL16188) [

20 ]. The genome assemblies were aligned to the BL16188 genome using Snippy v3.1 [ 30
]. Columns with gaps or ambiguous bases were masked, retaining a 1 863 980- bp core alignment (87% of BL16188) with 8403 polymorphic sites. Gubbins v1.4.1 (m = 5, i = 10, BL16188) [ 31
] was used to mask recombination events, and a final tree was produced using RAxML v8.2.9 with Stamatakis ascertainment bias correction and 600 bootstraps terminated by MRE bootstopping [ 32
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