Journal of Infection (2012) 64, 184e187

www.elsevierhealth.com/journals/jinf

Fatal Neisseria meningitidis serogroup X sepsisin immunocompromised patients in Spain. Virulenceof clinical isolates

Diego Vicente a,b, Olatz Esnal b, Emilio Perez-Trallero a,b,c,*

aMicrobiology Service and Reference Laboratory for Meningococcal Infections of the Basque Country,Hospital Donostia-Instituto Biodonostia, 20014 San Sebasti�an, SpainbBiomedical Research Centre Network for Respiratory Diseases (CIBERES), San Sebasti�an, Spainc Faculty of Medicine, University of the Basque Country, San Sebasti�an, Spain

Accepted 9 November 2011Available online 15 November 2011

KEYWORDSNeisseria meningitidisX;MLST;Epidemiology;Virulence;In vivo animal study;Invasive disease

* Corresponding author. Hospitalfax: þ34943007470.

E-mail address: mikrobiol@terra.e

0163-4453/$36 ª 2011 The British Infdoi:10.1016/j.jinf.2011.11.009

Summary Background: Neisseria meningitidis X (NmX) is a minority meningococcus ser-ogroup for which no vaccine is available. Huge meningococcus X outbreaks occurred in WestAfrica but developed countries have reported only sporadic cases. Two invasive fatal casesare presented.Methods: Isolates were characterized by serogrouping (latex agglutination and genogroupX-specific polymerase-chain-reaction), PorA and FetA typing, multilocus sequence typing andpulsed-field gel electrophoresis. Immunocompetent female balm/C mice inoculated intraper-itoneally were use to test virulence of invasive and carrier isolates.Results: Until April 2010, NmX was absent among 868 invasive meningococci characterized inthe Basque Country in a 20-year period. In April 2010, two fatal NmX: P1.21,16: F5-5/ST750episodes were detected in unrelated immunodeficient patients. After analysis of 803 meningo-coccal isolates from asymptomatic carriers obtained between 1988 and 2010, eight NmX iso-lates were detected. The genotype of the two invasive isolates bore no relation to any ofthe NmX isolates detected in healthy individuals from the Basque Country or to isolates fromoutbreaks in Africa.Conclusions: NmX isolates in the north of Spain can cause severe disease in humans, despitetheir low prevalence. The in vivo animal study showed that virulence of isolates was moreclosely associated with the genotype than with the serogroup.ª 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Donostia, Paseo Dr Beguiristain s/n, 20014 San Sebasti�an, Spain. Tel.: þ34943007046;

s (E. Perez-Trallero).

ection Association. Published by Elsevier Ltd. All rights reserved.

Meningococcus X causing invasive disease in Spain 185

Introduction

Neisseria meningitidis serogroup X (NmX) can cause sub-stantial morbidity and mortality and a vaccine is currentlylacking.1 Until recently, this serogroup was considered tohave low virulence and was usually detected as a pharyn-geal colonizer in healthy individuals.2

The prominence of this microorganism has increased dueto recent outbreaks in Africa, where local outbreaks3,4 andthree large-scale epidemics in Niger, Togo and BurkinaFaso5,6 have been reported with high morbidity and mortal-ity. In developed countries, sporadic episodes of meningo-coccal disease caused by NmX,2,7e9 whose geneticpatterns differed from those in Africa, have been reported.

In the spring of 2010 two fatalNmXepisodesweredetectedin the BasqueCountry, in northern Spain. The aimof this studywas to describe these cases and the in vivo virulence of theseisolates compared with that of other similar clones.

Materials and methods

The identification and genotypic characterization of in-vasive and carrier isolates were performed as previouslyreported.10 Briefly, the capsular serogroup was identifiedthrough latex agglutination and was confirmed by gen-ogroup X-specific real-time polymerase chain reaction(PCR).11 The genosubtype (PorA type) and FetA type weredetermined using the recommended typing targets pro-posed by the European Meningococcal Disease Society, se-quencing, and subsequent comparison of the deducedamino acid sequence in the N. meningitidis PorA and FetAvariable region database (http://pubmlst.org/neisseria/).

Multilocus sequence typing (MLST) was performed byamplification and sequencing of 7 housekeeping genes.Primers, determination of sequence alleles, and designa-tion of sequence types (ST) are described on the MLST website (http://pubmlst.org/neisseria/). Pulsed-field gel elec-trophoresis (PFGE) was performed according to previouslydescribed standard protocols12 using SpeI restriction endo-nuclease (Amersham Pharmacia Biotech, Buckinghamshire,England). The genotypic characteristics of isolates reportedin this study were deposited in the PubMLST Database.

The virulence of each isolate was studied using an animalmodel following directive 2003/65/EC of the European

Table 1 Date of isolation, source and typing schemes for charactin the Basque Country, northern Spain.

MLST IDa Date Source PorA VR1 PorA

17872 Apr-2010 Blood, invasive 21 1617875 Apr-2010 Blood, invasive 21 1617868 Jan-2005 Pharyngeal carrier 18 25e17870 Jan-2000 Pharyngeal carrier 22 1417869 Mar-2000 Pharyngeal carrier 21 1617873 Dec-1997 Pharyngeal carrier 7e30 3017867 Apr-1992 Pharyngeal carrier 12e1 13e17874 Jul-1992 Pharyngeal carrier 7e1 4e117871 Nov-1991 Pharyngeal carrier 19 15e17876 Mar-1989 Pharyngeal carrier 21 16a ID: Identification number of MLST database.

Parliament and the Spanish Royal Decree 1201/2005 on theprotection of animals used in experimentation. Immuno-competent female balm/C mice weighing 18e20 g wereinoculated intraperitoneally with 0.5 mL of a Neisseria men-ingitidis Mueller Hinton II suspension, containing106e107 CFU/mL. Mucine (M-2378, Sigma Chemical Co., St.Louis, MO) at a final concentration of 5% (wt/vol) was usedas adjuvant. Mortality over a 15-day follow-up period was re-corded daily. Six animals were used for each of the N. men-ingitidis isolates tested. Each experimental test wascontrolled with one untreated animal.

Results

In April 2010, 2 episodes of invasive meningococcal diseasedue to NmX were detected in Gipuzkoa (Northern Spain),with no apparent association between them, despite theshort time period between their occurrences. These werethe first 2 invasive episodes caused by NmX since surveil-lance was started in the meningococcus reference labora-tory of the Basque Country in 1988, where 868 invasivemeningococcal isolates were characterized up to 2010.The first of these 2 episodes occurred in a 27-year-oldman with a history of paroxysmal nocturnal hemoglobinuriaunder treatment with eculizumab (a monoclonal antibodythat targets complement fraction 5) and rivaroxaban (a fac-tor Xa inhibitor). The patient was vaccinated 16 months be-fore the episode of sepsis with the tetravalentmeningococcal polysaccharide vaccine (A, C, W135 andY), but the vaccine may not provide protection from menin-gococcal X infection and no antibiotic prophylaxis was doneduring treatment with eculizumab. On April 4, 2010 the pa-tient presented with high fever and breathlessness and de-veloped severe respiratory failure, generalized petechiaeand septic shock a few hours later. The patient died inthe intensive care unit (ICU) on the same day.

The second episode occurred in a 15-year-old girl, wholived 40 km away from the previous patient. The girl hadbeen diagnosed with systemic lupus erythematosus in 2005and was receiving mycophenolate (an immunosuppressorthat inhibits inosine monophosphate dehydrogenase) andoral prednisone (40 mg/day) and had previously beenvaccinated with the tetravalent meningococcal polysac-charide vaccine (A, C, W135 and Y). She did not receive

erization of the 10 serogroup X Neisseria meningitidis isolates

VR2 FetA MLST Clonal complex PFGE pattern

F5-5 750 ST-750 complex AF5-5 750 ST-750 complex A

14 e 823 ST-198 complex BF5-9 162 ST-162 complex CF3-6 8642 none DF1-5 3671 ST-53 complex E

1 F3-16 8641 none FF4-1 8644 none G

1 F5-2 8643 none HF3-16 8645 none I

Figure 1 Pulsed-field gel electrophoresis of two invasive iso-lates of Neisseria meningitidis X ST750 (lanes 10e11), one inva-sive isolate serogroup B ST750 (lane 12) and eight serogroup Xisolates from healthy carriers (lanes 2e9) after digestion withspeI. Lanes 1 and 13 are molecular size markers.

186 D. Vicente et al.

antibiotic prophylaxis during immunosuppressive therapy.The patient presented to the hospital with fever andabdominal pain. A few hours later, lowering of the levelof consciousness and abrupt hemodynamic deteriorationwere observed. The patient died in less than 24 h, despitesupport measures in the ICU. NmX was isolated in the bloodcultures of both patients.

Genetic analysis of the two invasive isolates revealed anidentical genotype in both: NmX:P1.21,16:F5-5:ST750(clonal complex: ST-750). Apart from these 2 isolates,only 1 new strain among all the invasive isolates in our col-lection belonged to this ST-750 complex, and was serogroupB (NmB:P1.21,16:F5-5:ST750).

After analysis of 803 meningococcal isolates fromasymptomatic carriers obtained between 1988 and 2010,eight NmX isolates were detected, none of which belonged

Table 2 Study of the virulence of 7 Neisseria meningitidis isinfection.

MLST IDa Source of isolates Serogroup MLST

17875 Blood, invasive X 75017872 Blood, invasive X 75018408 Blood, invasive B 7509550 Blood, invasive C 1117876 Pharyngeal carrier X 864517868 Pharyngeal carrier X 82317873 Pharyngeal carrier X 3671a ID: Identification number of MLST database.b ST8645 matches 6 out 7 alleles of ST5329, one isolate of the hype

to a clonal complex considered to be hypervirulent, al-though one of the isolates matched 6 out of 7 alleles ofa United Kingdom ST5329 isolate, belonging to the hypervir-ulent ST-269 clonal complex.

The genotype of the 2 invasive isolates bore no relationto any of the 8 NmX isolates detected from healthy individ-uals (Table 1). Table 1 includes broad molecular typingschemes for the characterization of the isolates, whichcould be useful in the potential development of “groupX” meningococcal vaccines. PFGE analysis showed an iden-tical pulsotype between the 2 invasive isolates and 8 dis-tinct pulsotypes among the 8 isolates from carriers(Fig. 1). In contrast, the serogroup B isolate belonging tothe ST-750 clonal complex showed high similarity (90%) tothe 2 invasive NmX isolates recently isolated.

For the animal model, 4 N. meningitidis invasive isolatesand 3 NmX isolates from carriers were selected. Of the in-vasive isolates, 3 ST750 (2 serogroup X and 1 serogroup B)and 1 from the hypervirulent ST11 serogroup C, whichserved as a control of extremely high virulence, were se-lected. Two of these 3 NmX isolates from pharyngeal car-riers belonged to clonal complexes whose members weremostly pharyngeal colonizer from healthy subjects. Thethird was the isolate close to the hypervirulent ST-269clonal complex (Table 2). Mortality in mice infected with2 of the isolates from carriers was nil but mortality fromthe third isolate was identical to that produced by the iso-late included as a hypervirulent control. Mortality producedby invasive isolates from the clonal complex ST-750,whether serogroup X or B, was intermediate.

Discussion

In developed countries, very few episodes of invasivemeningococcal disease are attributed to NmX and these ep-isodes frequently affect persons with risk factors,2 as in thetwo above-mentioned cases. In contrast, the presence of se-rogroup X is greater in developing countries, especially thosein the sub-Saharan belt, and has increased in the last fewyears. In addition to local outbreaks in Ghana3 and Kenia,4

there have been two major epidemics. The first occurredin 2006 in Niger where NmX caused more than 500 episodes,representing 51% of all confirmed meningococcal meningitiscases in that year.5 The second occurred between 2007 and2010 when NmX spread through Togo and Burkina Faso.6

olates in a mouse model of intraperitoneal meningococcal

ST-clonal complex Infected mice Dead mice (%)

750 6 3 (50)750 6 3 (50)750 6 2 (33)(ET37) ST11 6 6 (100)noneb 6 6 (100)198 6 0 (0)53 6 0 (0)

rvirulent ST-269 clonal complex.

Meningococcus X causing invasive disease in Spain 187

Genetic studies have shown that the NmX population ishighly diverse, grouping most of the strains causing invasivedisease in two, unrelated clonal groups.2 Clonal group XeIencompasses the invasive strains isolated in Africa, whilethe few European and American strains analyzed are group-ed in clonal group X-II (ST-750 complex). The two invasiveisolates isolated in our study belonged to this latter clonalgroup. The strains isolated in asymptomatic carriers haddistinct genotypes and PFGE patterns, and bore no relationto the strains isolated in healthy carriers in our environ-ment. NmX is infrequent in the Basque Country and wehad previously detected only one invasive isolate belongingto the ST-750 clonal complex, which was serogroup B.

The development of meningococcal disease depends onthe interaction between host-related factors and thestrain’s pathogenicity and virulence. In our experience, de-ficient immune status seems to be a determining factor forthe development of this disease. However, although lowervirulence has been attributed to serogroup X strains,2 twoisolates causing fatal episodes belonged to the ST-750clonal complex, which is a frequent cause of invasive epi-sodes (43% of the serogroup X isolates as found in theMLST database by July 2011were invasive). Meningococcalinfections are the most important adverse reactions experi-enced by patients receiving eculizumab therapy andthese patients might be at risk of disease by uncommonserogroups (particularly Y, W135 and X). http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/000791/WC500054208.pdf

Because the virulence of NmX isolates could have beenrecently altered, as suggested by the increase in the inci-dence of NmX invasive disease, we performed an in vitroanimal study comparing isolates from asymptomatic car-riers with the present two isolates causing severe disease.The results of the animal experiment performed in thisstudy suggest that the virulence of the isolate largely de-pends on components related to its internal structure inde-pendently of the serogroup or of the source of the isolate(patient or asymptomatic carrier).

Secondly, the results of this study suggest that, likeother isolates in Europe, NmX isolates in the north of Spainare sufficiently virulent to cause severe disease in humans,despite their low prevalence.

The foreseeable frequent use of conjugate meningococ-cal vaccine for serogroups A, C, Y and W135, with a powerfuleffect on pharyngeal carriage could favor the disseminationof serogroup X strains, whose virulence should not beunderestimated and against which there is currently novaccine.

Acknowledgements

The authors acknowledge the use of the Neisseria MLSTwebsite (http://pubmlst.org/neisseria/) developed byKeith Jolley and Man-Suen Chan and located at the OxfordUniversity. This work was supported in part by Departmentof Health, Basque Country, Spain (PI2006111043).

Conflict of interest

None declared.

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