BACKGROUND
neisseria meningitidis  is a commensal of the human upper respiratory tract, which occasionally causes meningitis and sepsis, particularly in infants and young adults.
meningococci may express one of twelve antigenically distinct capsules, however, invasive disease is mostly caused by serogroups a, b, c, w, x or y. the polysaccharide capsule is an important virulence determinant and aids in evading the host immune response  <cit> .
enzymes for capsule biosynthesis and transport are encoded by a single cluster of genes termed the capsular polysaccharide synthesis  locus, which is divided into six regions arranged in the order of d-a-c-e-d’-b  <cit> .
polysaccharide synthesis is encoded by region a genes which vary according to serogroup, whilst regions b  and c  are responsible for capsular transport  <cit> .
region d  is involved in lipooligosaccharide biosynthesis and d’ , a truncated copy of region d, is non-functional  <cit> .
region e is not involved in polysaccharide synthesis or transport, but is hypothesized to regulate these processes  <cit> .
loss of capsule expression may be caused by horizontal genetic exchange, slipped-strand mispairing, point mutations or gene deletion in the cps locus  <cit> .
meningococci which lack cps genes are regarded as non-pathogenic.
however, rare cases of invasive disease caused by meningococci lacking regions a, b and c of the cps locus and containing a capsule null locus , have been reported in germany, canada, burkina faso and china .
twenty-six cnl alleles  were defined in the pubmlst neisseria spp.
database  at the time of this study, with some alleles present in other neisseria including n. lactamica and n. gonorrhoeae  <cit> .
genetic lineages described for invasive cnl isolates include those belonging to sequence type  st- <dig>  192), st- <dig>  and st- <dig>   <cit> .
clonal complex  <dig> isolates are typically associated with carriage  <cit>  although invasive isolates have been documented on the pubmlst neisseria database.
polysaccharide-based vaccines that target serogroups a, c, w, and y are ineffective against cnl strains however, protein-based meningococcal vaccines developed for serogroup b, such as bexsero® and the bivalent factor h-binding protein  vaccine trumenba®, have the potential to target non-serogroup b including cnl meningococci .
through national laboratory-based surveillance for invasive meningococcal disease  from  <dig> through  <dig>  we identified four cnl meningococci.
we aimed to describe the respective cases and characterize the isolates using whole genome data.
RESULTS
identification and characterization of invasive capsule null meningococci
from  <dig> through  <dig>   <dig> cases of imd were reported, with viable isolates available for  <dig>  cases.
we identified five n. meningitidis isolates that were phenotypically and genotypically negative for serogroups a, b, c, w, x and y. the isolates were also ctra pcr negative but were sodc pcr positive.
transmission electron microscopy  confirmed the absence of a polysaccharide capsule .fig.
<dig> transmission electron micrographs showing the presence of surface capsular polysaccharide for  neisseria meningitidis serogroup w  and  absence of capsule for neisseria lactamica .
clinical isolates from south africa are depicted in   <dig>   <dig>   <dig> and   <dig>  the scale bar represent  <dig> nm
one of the five isolates,  <dig>  did not contain a cnl allele, belonged to st- <dig>  and contained capsule transport genes in region b  and lipopolysaccharide synthesis genes  in region d of the cps locus.
all region a genes except for ctrg were absent, as well as ctra from region c. the remaining four ctra negative isolates lacked genes in regions a and c  and contained a 114bp cnl allele.
further, these isolates contained all region d lipopolysaccharide synthesis genes and lacked region b genes.
the locus was identified as cnl allele  <dig> in isolate  <dig>  and cnl allele  <dig> in isolates  <dig>   <dig> and  <dig>   <cit> .
isolates  <dig>   <dig> and  <dig> lacked the feta locus.
the finetypes for the four cnl isolates were as follows: isolate  <dig> was ng: p <dig> - <dig> : f1-2: st- <dig> , and isolates  <dig>   <dig> and  <dig> were ng: p <dig> - <dig> -2: fΔ: st- <dig> .
clinical case descriptions
the four invasive cnl meningococi were isolated from three patients, including one patient with recurrent meningococcal disease .
all three patients responded well to antibiotic therapy and subsequently recovered from their imd episodes.
the four cnl isolates were susceptible to all antimicrobials except for trimethoprim-sulfamethoxazole.
additional information regarding vaccination status and long-term complications for all three patients was sought, but unfortunately these data were not available.table  <dig> patient demographic information and phenotypic and genotypic characteristics of four invasive capsule null neisseria meningitidis isolates identified through national laboratory-based surveillance in south africa, 2003–2013
abbreviations: copd chronic obstructive pulmonary disease, c <dig> sixth complement component, csf cerebrospinal fluid, s susceptible, r resistant, ng non-groupable, p <dig> pora, f feta, st sequence type, cc clonal complex, Δ gene deletion
apatient three presented with two episodes of invasive meningococcal disease in  <dig>  and  <dig> , respectively.
he was diagnosed with deficiency of the sixth complement component
the first patient, an adult male, was previously diagnosed with multiple chronic illnesses including diabetes mellitus, hypertension, osteoarthritis and chronic obstructive pulmonary disease.
he was a smoker, morbidly obese and had a right lower lobe lobectomy in  <dig> due to damage from a previous tuberculosis infection.
in  <dig>  he was diagnosed with empyema and n. meningitidis was cultured from the pleural fluid .
the second patient, an adolescent male, was diagnosed with meningococcal meningitis in  <dig> .
unfortunately, information regarding underlying disease conditions and the severity of disease could not be obtained.
the third patient was a male child with deficiency of the sixth complement component .
in  <dig>  he was diagnosed with meningitis , however,  <dig> months later, in  <dig>  he presented with fever and disorientation and n. meningitidis was isolated from the blood .
he was prescribed life-long treatment with penicillin.
his mother received a dose of the quadrivalent conjugate vaccine  in  <dig>
comparison of south african and other capsule null meningococcal genomes
the four south african  isolates were compared to  <dig> cnl meningococcal genomes available on the pubmlst neisseria database, including four cc <dig> burkina faso isolates that were sequenced as part of this study .
the remaining  <dig> isolates belonged to seven clonal complexes including cc <dig> , cc <dig> , cc41/ <dig> , cc <dig> , cc <dig> , cc <dig>  and cc <dig> .
all cnl meningococci  harboured fhbp and nhba, but lacked nada.
phylogenetic analysis of  <dig> ribosomal mlst  loci clustered the  <dig> cnl isolates by clonal complex .
data for  <dig> of  <dig> core genome mlst  loci were incomplete and were excluded from further analysis.
phylogenetic analysis of the remaining  <dig> of  <dig> cgmlst loci in all cnl isolates , demonstrated clustering by clonal complex .
isolates within each respective clonal complex contained identical cnl alleles, except for cc41/ <dig> which contained alleles  <dig> or 12; and cc <dig> which contained alleles  <dig> or  <dig>  additional analysis of  <dig> putative virulence loci in  <dig> carriage and  <dig> invasive cnl isolates including those from south africa, determined no mutually exclusive loci or alleles .
most putative virulence loci were identified in both carriage and invasive isolates , and the remaining  <dig> loci were absent in all  <dig> isolates.fig.
<dig> phylogenetic analysis of  <dig> of  <dig> core genes  genes in capsule null neisseria meningitidis isolates  belonging to clonal complexes   <dig> , cc <dig> , cc <dig> , cc <dig> , cc41/ <dig> , cc <dig>  and cc <dig> .
individual cgmlst phylogenies are also illustrated for cc <dig> and cc <dig>  clusters are highlighted in grey and the invasive south african isolates are represented by red nodes.
isolates  <dig> and  <dig> were from recurrent invasive disease episodes in the same patient.
the scale bars represent the number of variant loci.
all genomes are available on the http://pubmlst.org/neisseria website
clonal complex 53
at the time of analysis, in addition to isolate  <dig>  complete genome data were available for  <dig> non-groupable cc <dig> isolates in the pubmlst neisseria database.
forty-seven were carriage isolates from either the uk , czech republic , italy  or germany , and one was an invasive isolate from ireland in  <dig>  the cc <dig> isolates  belonged to one of six sts, namely, st- <dig> , st- <dig> , st- <dig> , st- <dig> , st- <dig>  or st- <dig> .
all cc <dig> isolates harboured cnl allele  <dig> flanked by gale allele  <dig>  and one of five tex alleles .
gale allele  <dig> was present in one cc41/ <dig> isolate, also harbouring cnl allele  <dig>  all cc <dig> isolates possessed alleles  <dig> or  <dig> for the fhbp family 2/subfamily a antigen.
the nhba allele  <dig>  was present in all of the cc <dig> isolates, except the sa isolate which harboured allele  <dig> .
using seven-locus mlst, the cc <dig> isolates were resolved into six clusters .
ribosomal mlst further resolved these isolates into  <dig> clusters and the sa isolate had a unique rmlst profile .
core genome mlst indicated that the sa isolate was more closely related to two carriage isolates circulating in the czech republic in  <dig>  than to carriage isolates from the uk, italy and germany, and the invasive isolate from ireland .
overall, 221/ <dig>  cgmlst loci had identical nucleotide sequences amongst the cc <dig> isolates .
clonal complex 192
genome data were available in the pubmlst neisseria database for four burkina faso isolates sequenced as part of this study, and six carriage isolates from malawi.
analysis of the cps locus of the burkina faso isolates confirmed the absence of regions a, b and c and the presence of cnl allele  <dig> flanked by gale allele  <dig> and tex allele  <dig>  <cit> .
in contrast, the sa and malawi isolates harboured cnl allele  <dig> flanked by gale allele  <dig> and tex allele  <dig>  all gale and tex alleles were unique to cc <dig> cnl isolates, and were not found in any other clonal complex nor in encapsulated isolates in the pubmlst neisseria database.
the burkina faso isolates were finetype ng: p <dig> - <dig> : fΔ: st- <dig>  whilst the sa and malawi isolates were ng: p <dig> - <dig> -2: fΔ: st- <dig> .
all  <dig> isolates harboured allele  <dig> for the fhbp family 3/subfamily a antigen and lacked both the feta and nada loci.
eleven of  <dig> isolates had allele  <dig> for nhba peptide  <dig>
ribosomal mlst and cgmlst resolved the  <dig> cc <dig> isolates into three groups, which were congruent with country of origin.
the sa and malawi isolates were more closely related to each other , compared to the burkina faso isolates, which differed from the sa meningococci by  <dig>  loci and the malawi meningococci by  <dig>  loci, respectively  .
isolate  <dig> from patient two, differed by  <dig>  and  <dig> cgmlst loci  from isolates  <dig> and  <dig> which were from recurrent imd episodes in patient three, respectively.
isolates  <dig> and  <dig> differed from each other by two cgmlst metabolic loci  .
further nucleotide analysis indicated that both genes differed by one nucleotide.
overall, 660/ <dig>  of cgmlst loci shared identical nucleotide sequences amongst the  <dig> cc <dig> isolates.
discussion
four invasive cnl meningococci were detected from three patients, including recurrent imd in a c <dig> complement deficient patient.
there was no epidemiological link between patients, and one isolate was ng: p <dig> - <dig> : f1-2: st- <dig>  whilst the remaining three were ng: p <dig> - <dig> -2: fΔ: st- <dig> .
these genotypes were different to invasive cnl isolates reported in other countries .
one invasive isolate did not contain a cnl allele, but lacked most genes from regions a and c of the cps.
this was similar to that described previously in a non-groupable cc41/ <dig> carriage isolate from the usa  <cit> .
meningococci of cc <dig> with cnl have previously been found in 7% of n. meningitidis isolates from healthy carriers in germany  <cit> .
meningococcal carriage data from the african meningitis belt suggest that cc <dig> is not common in this region  with searches in the pubmlst neisseria database identifying only three other non-groupable cc <dig> carriage isolates from this region: two from senegal and one in ethiopia.
in addition to an invasive irish isolate which possessed the same finetype as the south african isolate,  <dig> other invasive non-groupable cc <dig> isolates were identified in cuba, cyprus, france and the uk, of which six isolates were ng: p <dig> ,30: f-nd: st- <dig> .
carriage rates are currently unknown in south africa and we have no knowledge of carried genotypes.
although we do not have genotypic data for all of our invasive isolates the earliest documentation of any cc <dig> strain in south africa is the cnl isolate identified in  <dig>  and described in this study.
in contrast to cc <dig>  cc <dig> has been reported among carriers in countries in the african meningitis belt including burkina faso  <cit> , ghana  <cit> , the gambia, ethiopia, mali, uganda and niger .
according to the pubmlst neisseria database, 95%  of cc <dig> isolates were isolated in africa, and 4%  isolated in europe; as opposed to carriage cc <dig> isolates which were predominantly isolated in europe  and rarely observed in africa .
furthermore, two cc <dig> carriage isolates were documented in norway and sweden, and one case of bacteremia was documented in france .
the swedish and french isolates shared the same strain designation as the south african and malawi isolates, however genomic data were unavailable for these isolates.
three cases of invasive disease caused by cnl st- <dig> isolates occurred in burkina faso in  <dig> and  <dig>  two of which were included in our study along with two carriage isolates from  <dig>  <cit> .
core genome mlst analysis of  <dig> african isolates revealed three groups of cc <dig> isolates clustering according to their respective countries, however the dataset in this study was limited and more cc <dig> genomes would be required to validate this geographic clustering and fully describe the molecular epidemiology of this clone.
the lack of a polysaccharide capsule in disease-associated isolates implies that factors other than encapsulation may contribute to the ability of a strain to cause invasive disease, including underlying disease conditions of the host.
disease due to unencapsulated meningococci and recurrent imd, have been described in immunocompromised patients who are deficient in terminal pathway complement proteins c <dig> through c <dig> .
in our study, one patient with c <dig> deficiency had recurrent imd with the same cnl strain.
a similar case was described in the usa in a 5-month-old male who was also diagnosed with c <dig> deficiency  <cit> .
he was first diagnosed with meningitis followed by bacteremia six months later.
both episodes were likely to have been caused by the same unencapsulated meningococcal strain, based on the fact that the isolates were non-groupable by phenotypic serogrouping and had identical outer membrane vesicle profiles on sds-page, however additional genotypic data were not available for confirmation.
these cases of recurrent imd may suggest persistent carriage in close contacts which is further supported by mueller et al.
<cit>  who identified non-groupable cc <dig> meningococci at three consecutive monthly visits in six healthy carriers in burkina faso.
in south africa, chemoprophylaxis is recommended for close contacts of imd patients to eradicate carriage, however this particular strain may have persisted within the family.
this does not however exclude the possibility that the second isolate may have been re-acquired from an individual in the community.
philadelphia chromosome-positive  common acute lymphatic leukemia was reported in a patient with imd caused by a st- <dig>  cnl isolate in germany, in  <dig>  <cit> .
in our study, the st- <dig>  isolate was the only organism to be cultured from pleural fluid of a patient with empyema, indicating that this isolate was the most likely cause of invasive disease.
further, imd by the st- <dig>  isolate occurred in a patient who was immunocompromised due to diabetes mellitus and, in addition, presented with multiple chronic illnesses which may have contributed to his susceptibility to invasive disease with a cnl strain.
molecular epidemiology and previous data from serum bactericidal assays  suggest that some groups of meningococci are more inclined to cause invasive disease than others, with encapsulated strains being more resistant to complement killing than their unencapsulated counterparts  <cit> .
although the polysaccharide capsule has been shown to be an important virulence determinant, previous sba data indicate that the ability of the invasive cnl st- <dig>  isolates from burkina faso to resist complement killing in normal human sera, was comparable to an encapsulated serogroup b strain  <cit> .
the invasive st- <dig>  isolates from burkina faso were also determined to be more resistant to complement killing than a carriage st- <dig>  isolate and an invasive st- <dig>  isolate, which had similar resistance profiles to each other and to an unencapsulated serogroup b mutant.
exogenous lipooligosaccharide sialylation significantly increased resistance to complement killing in two invasive cnl isolates belonging to st- <dig> , and was partially attributed to their ability to cause invasive disease in apparently healthy patients  <cit> .
however, this mechanism was not identified in the invasive st- <dig>  isolates from burkina faso, the carriage st- <dig>  isolate and the invasive st- <dig>  isolate  <cit> .
in agreement with previous genome studies, most putative virulence loci were present in both carriage and invasive cnl isolates .
although joseph et al.
<cit>  determined significant associations of mobile genetic elements with invasive meningococci, their contribution to meningococcal virulence is unknown.
the ability of cnl isolates to cause invasive disease may likely be due to host risk factors, however differences in the virulence potential may also be explained by variation in gene expression.
predisposing factors for one patient with invasive disease that was caused by an st- <dig>  isolate, were unknown.
it is likely that this patient may have also presented with underlying disease, however sba data for the invasive st- <dig>  isolates from burkina faso, indicate that these isolates may cause invasive disease in healthy patients.
in contrast, cc <dig> which was previously shown to be sensitive to normal human sera, may require an immunocompromised host to cause imd, as demonstrated in our study.
we did not perform serum bactericidal assays to confirm these findings.
in south africa, the quadrivalent polysaccharide vaccine and the quadrivalent conjugate vaccine which was recently introduced in  <dig>  are recommended for individuals with terminal complement deficiencies and may be offered to close contacts of imd patients following post-exposure chemoprophylaxis.
the vaccination status of all three patients as well as the contacts for the first two were unknown, however the mother of the patient with c <dig> deficiency was administered a single dose of menactra®, which is ineffective in preventing carriage of cnl meningococci which lack a capsule.
all  <dig> cnl meningococci including those analyzed in this study lacked the nada locus and the p <dig>  antigen; and most isolates expressed fhbp variants which are not targeted by the bexsero® vaccine .
the effectiveness of bexsero® to target cnl meningococci in general would therefore be largely reliant on the expression and cross protective potential of nhba  <cit> .
although the bivalent fhbp vaccine trumenba® potentially elicits broad spectrum bactericidal activity against serogroup b strains, its effect on fhbp variants and their level of expression in other serogroups and cnl meningococci is unknown  <cit> .
CONCLUSIONS
invasive meningococcal disease by cnl meningococci in south africa is rare however such strains may have a heightened tendency to cause imd in an immunocompromised host, potentially coupled with currently unknown non-capsular virulence mechanisms in the meningococcus.

 neisseria meningitidis is a commensal of the human upper respiratory tract, which occasionally causes meningitis and sepsis, particularly in infants and young adults. meningococci may express one of twelve antigenically distinct capsules, however, invasive disease is mostly caused by serogroups a, b, c, w, x or y. the polysaccharide capsule is an important virulence determinant and aids in evading the host immune response. enzymes for capsule biosynthesis and transport are encoded by a single cluster of genes termed the capsular polysaccharide synthesis locus, which is divided into six regions arranged in the order of d-a-c-e-d’-b. polysaccharide synthesis is encoded by region a genes which vary according to serogroup, whilst regions b and c are responsible for capsular transport. region d is involved in lipooligosaccharide biosynthesis and d’, a truncated copy of region d, is non-functional. region e is not involved in polysaccharide synthesis or transport, but is hypothesized to regulate these processes. loss of capsule expression may be caused by horizontal genetic exchange, slipped-strand mispairing, point mutations or gene deletion in the cps locus. meningococci which lack cps genes are regarded as non-pathogenic. however, rare cases of invasive disease caused by meningococci lacking regions a, b and c of the cps locus and containing a capsule null locus, have been reported in germany, canada, burkina faso and china. twenty-six cnl alleles were defined in the pubmlst neisseria spp. database at the time of this study, with some alleles present in other neisseria including n. lactamica and n. gonorrhoeae. genetic lineages described for invasive cnl isolates include those belonging to sequence type st- 192), st- and st-. clonal complex isolates are typically associated with carriage although invasive isolates have been documented on the pubmlst neisseria database. polysaccharide-based vaccines that target serogroups a, c, w, and y are ineffective against cnl strains however, protein-based meningococcal vaccines developed for serogroup b, such as bexsero® and the bivalent factor h-binding protein vaccine trumenba®, have the potential to target non-serogroup b including cnl meningococci. through national laboratory-based surveillance for invasive meningococcal disease from through we identified four cnl meningococci. we aimed to describe the respective cases and characterize the isolates using whole genome data.  identification and characterization of invasive capsule null meningococci from through cases of imd were reported, with viable isolates available for cases. we identified five n. meningitidis isolates that were phenotypically and genotypically negative for serogroups a, b, c, w, x and y. the isolates were also ctra pcr negative but were sodc pcr positive. transmission electron microscopy confirmed the absence of a polysaccharide capsule.fig. transmission electron micrographs showing the presence of surface capsular polysaccharide for neisseria meningitidis serogroup w and absence of capsule for neisseria lactamica. clinical isolates from south africa are depicted in and the scale bar represent nm one of the five isolates, did not contain a cnl allele, belonged to st- and contained capsule transport genes in region b and lipopolysaccharide synthesis genes in region d of the cps locus. all region a genes except for ctrg were absent, as well as ctra from region c. the remaining four ctra negative isolates lacked genes in regions a and c and contained a 114bp cnl allele. further, these isolates contained all region d lipopolysaccharide synthesis genes and lacked region b genes. the locus was identified as cnl allele in isolate and cnl allele in isolates and. isolates and lacked the feta locus. the finetypes for the four cnl isolates were as follows: isolate was ng: p -: f1-2: st-, and isolates and were ng: p - -2: fΔ: st-. clinical case descriptions the four invasive cnl meningococi were isolated from three patients, including one patient with recurrent meningococcal disease. all three patients responded well to antibiotic therapy and subsequently recovered from their imd episodes. the four cnl isolates were susceptible to all antimicrobials except for trimethoprim-sulfamethoxazole. additional information regarding vaccination status and long-term complications for all three patients was sought, but unfortunately these data were not available.table patient demographic information and phenotypic and genotypic characteristics of four invasive capsule null neisseria meningitidis isolates identified through national laboratory-based surveillance in south africa, 2003–2013     abbreviations: copd chronic obstructive pulmonary disease, c sixth complement component, csf cerebrospinal fluid, s susceptible, r resistant, ng non-groupable, p pora, f feta, st sequence type, cc clonal complex, Δ gene deletion apatient three presented with two episodes of invasive meningococcal disease in and, respectively. he was diagnosed with deficiency of the sixth complement component the first patient, an adult male, was previously diagnosed with multiple chronic illnesses including diabetes mellitus, hypertension, osteoarthritis and chronic obstructive pulmonary disease. he was a smoker, morbidly obese and had a right lower lobe lobectomy in due to damage from a previous tuberculosis infection. in he was diagnosed with empyema and n. meningitidis was cultured from the pleural fluid. the second patient, an adolescent male, was diagnosed with meningococcal meningitis in. unfortunately, information regarding underlying disease conditions and the severity of disease could not be obtained. the third patient was a male child with deficiency of the sixth complement component. in he was diagnosed with meningitis, however, months later, in he presented with fever and disorientation and n. meningitidis was isolated from the blood. he was prescribed life-long treatment with penicillin. his mother received a dose of the quadrivalent conjugate vaccine in comparison of south african and other capsule null meningococcal genomes the four south african isolates were compared to cnl meningococcal genomes available on the pubmlst neisseria database, including four cc burkina faso isolates that were sequenced as part of this study. the remaining isolates belonged to seven clonal complexes including cc, cc, cc41/, cc, cc, cc and cc. all cnl meningococci harboured fhbp and nhba, but lacked nada. phylogenetic analysis of ribosomal mlst loci clustered the cnl isolates by clonal complex. data for of core genome mlst loci were incomplete and were excluded from further analysis. phylogenetic analysis of the remaining of cgmlst loci in all cnl isolates, demonstrated clustering by clonal complex. isolates within each respective clonal complex contained identical cnl alleles, except for cc41/ which contained alleles or 12; and cc which contained alleles or additional analysis of putative virulence loci in carriage and invasive cnl isolates including those from south africa, determined no mutually exclusive loci or alleles. most putative virulence loci were identified in both carriage and invasive isolates, and the remaining loci were absent in all isolates.fig. phylogenetic analysis of of core genes genes in capsule null neisseria meningitidis isolates belonging to clonal complexes, cc, cc, cc, cc41/, cc and cc. individual cgmlst phylogenies are also illustrated for cc and cc clusters are highlighted in grey and the invasive south african isolates are represented by red nodes. isolates and were from recurrent invasive disease episodes in the same patient. the scale bars represent the number of variant loci. all genomes are available on the http://pubmlst.org/neisseria website clonal complex 53 at the time of analysis, in addition to isolate complete genome data were available for non-groupable cc isolates in the pubmlst neisseria database. forty-seven were carriage isolates from either the uk, czech republic, italy or germany, and one was an invasive isolate from ireland in the cc isolates belonged to one of six sts, namely, st-, st-, st-, st-, st- or st-. all cc isolates harboured cnl allele flanked by gale allele and one of five tex alleles. gale allele was present in one cc41/ isolate, also harbouring cnl allele all cc isolates possessed alleles or for the fhbp family 2/subfamily a antigen. the nhba allele was present in all of the cc isolates, except the sa isolate which harboured allele. using seven-locus mlst, the cc isolates were resolved into six clusters. ribosomal mlst further resolved these isolates into clusters and the sa isolate had a unique rmlst profile. core genome mlst indicated that the sa isolate was more closely related to two carriage isolates circulating in the czech republic in than to carriage isolates from the uk, italy and germany, and the invasive isolate from ireland. overall, 221/ cgmlst loci had identical nucleotide sequences amongst the cc isolates. clonal complex 192 genome data were available in the pubmlst neisseria database for four burkina faso isolates sequenced as part of this study, and six carriage isolates from malawi. analysis of the cps locus of the burkina faso isolates confirmed the absence of regions a, b and c and the presence of cnl allele flanked by gale allele and tex allele. in contrast, the sa and malawi isolates harboured cnl allele flanked by gale allele and tex allele all gale and tex alleles were unique to cc cnl isolates, and were not found in any other clonal complex nor in encapsulated isolates in the pubmlst neisseria database. the burkina faso isolates were finetype ng: p -: fΔ: st- whilst the sa and malawi isolates were ng: p - -2: fΔ: st-. all isolates harboured allele for the fhbp family 3/subfamily a antigen and lacked both the feta and nada loci. eleven of isolates had allele for nhba peptide ribosomal mlst and cgmlst resolved the cc isolates into three groups, which were congruent with country of origin. the sa and malawi isolates were more closely related to each other, compared to the burkina faso isolates, which differed from the sa meningococci by loci and the malawi meningococci by loci, respectively. isolate from patient two, differed by and cgmlst loci from isolates and which were from recurrent imd episodes in patient three, respectively. isolates and differed from each other by two cgmlst metabolic loci. further nucleotide analysis indicated that both genes differed by one nucleotide. overall, 660/ of cgmlst loci shared identical nucleotide sequences amongst the cc isolates. discussion four invasive cnl meningococci were detected from three patients, including recurrent imd in a c complement deficient patient. there was no epidemiological link between patients, and one isolate was ng: p -: f1-2: st- whilst the remaining three were ng: p - -2: fΔ: st-. these genotypes were different to invasive cnl isolates reported in other countries. one invasive isolate did not contain a cnl allele, but lacked most genes from regions a and c of the cps. this was similar to that described previously in a non-groupable cc41/ carriage isolate from the usa. meningococci of cc with cnl have previously been found in 7% of n. meningitidis isolates from healthy carriers in germany. meningococcal carriage data from the african meningitis belt suggest that cc is not common in this region with searches in the pubmlst neisseria database identifying only three other non-groupable cc carriage isolates from this region: two from senegal and one in ethiopia. in addition to an invasive irish isolate which possessed the same finetype as the south african isolate, other invasive non-groupable cc isolates were identified in cuba, cyprus, france and the uk, of which six isolates were ng: p,30: f-nd: st-. carriage rates are currently unknown in south africa and we have no knowledge of carried genotypes. although we do not have genotypic data for all of our invasive isolates the earliest documentation of any cc strain in south africa is the cnl isolate identified in and described in this study. in contrast to cc cc has been reported among carriers in countries in the african meningitis belt including burkina faso, ghana, the gambia, ethiopia, mali, uganda and niger. according to the pubmlst neisseria database, 95% of cc isolates were isolated in africa, and 4% isolated in europe; as opposed to carriage cc isolates which were predominantly isolated in europe and rarely observed in africa. furthermore, two cc carriage isolates were documented in norway and sweden, and one case of bacteremia was documented in france. the swedish and french isolates shared the same strain designation as the south african and malawi isolates, however genomic data were unavailable for these isolates. three cases of invasive disease caused by cnl st- isolates occurred in burkina faso in and two of which were included in our study along with two carriage isolates from. core genome mlst analysis of african isolates revealed three groups of cc isolates clustering according to their respective countries, however the dataset in this study was limited and more cc genomes would be required to validate this geographic clustering and fully describe the molecular epidemiology of this clone. the lack of a polysaccharide capsule in disease-associated isolates implies that factors other than encapsulation may contribute to the ability of a strain to cause invasive disease, including underlying disease conditions of the host. disease due to unencapsulated meningococci and recurrent imd, have been described in immunocompromised patients who are deficient in terminal pathway complement proteins c through c. in our study, one patient with c deficiency had recurrent imd with the same cnl strain. a similar case was described in the usa in a 5-month-old male who was also diagnosed with c deficiency. he was first diagnosed with meningitis followed by bacteremia six months later. both episodes were likely to have been caused by the same unencapsulated meningococcal strain, based on the fact that the isolates were non-groupable by phenotypic serogrouping and had identical outer membrane vesicle profiles on sds-page, however additional genotypic data were not available for confirmation. these cases of recurrent imd may suggest persistent carriage in close contacts which is further supported by mueller et al. who identified non-groupable cc meningococci at three consecutive monthly visits in six healthy carriers in burkina faso. in south africa, chemoprophylaxis is recommended for close contacts of imd patients to eradicate carriage, however this particular strain may have persisted within the family. this does not however exclude the possibility that the second isolate may have been re-acquired from an individual in the community. philadelphia chromosome-positive common acute lymphatic leukemia was reported in a patient with imd caused by a st- cnl isolate in germany, in. in our study, the st- isolate was the only organism to be cultured from pleural fluid of a patient with empyema, indicating that this isolate was the most likely cause of invasive disease. further, imd by the st- isolate occurred in a patient who was immunocompromised due to diabetes mellitus and, in addition, presented with multiple chronic illnesses which may have contributed to his susceptibility to invasive disease with a cnl strain. molecular epidemiology and previous data from serum bactericidal assays suggest that some groups of meningococci are more inclined to cause invasive disease than others, with encapsulated strains being more resistant to complement killing than their unencapsulated counterparts. although the polysaccharide capsule has been shown to be an important virulence determinant, previous sba data indicate that the ability of the invasive cnl st- isolates from burkina faso to resist complement killing in normal human sera, was comparable to an encapsulated serogroup b strain. the invasive st- isolates from burkina faso were also determined to be more resistant to complement killing than a carriage st- isolate and an invasive st- isolate, which had similar resistance profiles to each other and to an unencapsulated serogroup b mutant. exogenous lipooligosaccharide sialylation significantly increased resistance to complement killing in two invasive cnl isolates belonging to st-, and was partially attributed to their ability to cause invasive disease in apparently healthy patients. however, this mechanism was not identified in the invasive st- isolates from burkina faso, the carriage st- isolate and the invasive st- isolate. in agreement with previous genome studies, most putative virulence loci were present in both carriage and invasive cnl isolates. although joseph et al. determined significant associations of mobile genetic elements with invasive meningococci, their contribution to meningococcal virulence is unknown. the ability of cnl isolates to cause invasive disease may likely be due to host risk factors, however differences in the virulence potential may also be explained by variation in gene expression. predisposing factors for one patient with invasive disease that was caused by an st- isolate, were unknown. it is likely that this patient may have also presented with underlying disease, however sba data for the invasive st- isolates from burkina faso, indicate that these isolates may cause invasive disease in healthy patients. in contrast, cc which was previously shown to be sensitive to normal human sera, may require an immunocompromised host to cause imd, as demonstrated in our study. we did not perform serum bactericidal assays to confirm these findings. in south africa, the quadrivalent polysaccharide vaccine and the quadrivalent conjugate vaccine which was recently introduced in are recommended for individuals with terminal complement deficiencies and may be offered to close contacts of imd patients following post-exposure chemoprophylaxis. the vaccination status of all three patients as well as the contacts for the first two were unknown, however the mother of the patient with c deficiency was administered a single dose of menactra®, which is ineffective in preventing carriage of cnl meningococci which lack a capsule. all cnl meningococci including those analyzed in this study lacked the nada locus and the p antigen; and most isolates expressed fhbp variants which are not targeted by the bexsero® vaccine. the effectiveness of bexsero® to target cnl meningococci in general would therefore be largely reliant on the expression and cross protective potential of nhba. although the bivalent fhbp vaccine trumenba® potentially elicits broad spectrum bactericidal activity against serogroup b strains, its effect on fhbp variants and their level of expression in other serogroups and cnl meningococci is unknown.  invasive meningococcal disease by cnl meningococci in south africa is rare however such strains may have a heightened tendency to cause imd in an immunocompromised host, potentially coupled with currently unknown non-capsular virulence mechanisms in the meningococcus. 
one isolate contained cnl allele and caused empyema in an adult male with bronchiectasis from tuberculosis, diabetes mellitus and a smoking history. we identified four invasive cnl meningococci through laboratory-based surveillance in south africa from through which we aimed to characterize using whole genome data. although rare, invasive disease by cnl meningococci may be associated with host immunodeficiency and such patients may benefit from protein-based meningococcal vaccines. capsule null locusinvasive diseaseneisseria meningitidisafricast-53st-192national health laboratory service research trust 94442du plessis mignon bill and melinda gates foundation 307494010064512ganesh karistha http://dx.doi.org/ /100004440wellcome trust 087622http://dx.doi.org/ /100004440wellcome trust104992issue-copyright-statement© the author 2017 the remaining two isolates were from recurrent disease episodes in a male child with deficiency of the sixth complement component, and with the exception of two single nucleotide polymorphisms, contained identical core genomes. rare cases of invasive meningococcal disease caused by cnl isolates belonging to sequence types and clonal complexes st-, st-, st- and st- have been documented. the meningococcal capsule is an important virulence determinant. comparison of the south african genomes to additional cnl genomes on the pubmlst neisseria database, determined that most putative virulence genes could be found in both invasive and carriage phenotypes. unencapsulated meningococci lacking capsule biosynthesis genes and containing the capsule null locus are predominantly non-pathogenic. three isolates were ng: p - -2: fΔ: st- and contained cnl allele one patient was an adolescent male with meningitis.
 the meningococcal capsule is an important virulence determinant. unencapsulated meningococci lacking capsule biosynthesis genes and containing the capsule null locus are predominantly non-pathogenic. rare cases of invasive meningococcal disease caused by cnl isolates belonging to sequence types and clonal complexes st-, st-, st- and st- have been documented. the clinical significance of these isolates however remains unclear. we identified four invasive cnl meningococci through laboratory-based surveillance in south africa from through which we aimed to characterize using whole genome data.  one isolate contained cnl allele and caused empyema in an adult male with bronchiectasis from tuberculosis, diabetes mellitus and a smoking history. three isolates were ng: p - -2: fΔ: st- and contained cnl allele one patient was an adolescent male with meningitis. the remaining two isolates were from recurrent disease episodes in a male child with deficiency of the sixth complement component, and with the exception of two single nucleotide polymorphisms, contained identical core genomes. the st- isolate possessed alleles for nhba peptide and fhbp variant 2; whilst the st- isolates contained nhba peptide and fhbp variant all four isolates lacked nada. comparison of the south african genomes to additional cnl genomes on the pubmlst neisseria database, determined that most putative virulence genes could be found in both invasive and carriage phenotypes.  although rare, invasive disease by cnl meningococci may be associated with host immunodeficiency and such patients may benefit from protein-based meningococcal vaccines. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords capsule null locusinvasive diseaseneisseria meningitidisafricast-53st-192national health laboratory service research trust 94442du plessis mignon bill and melinda gates foundation 307494010064512ganesh karistha http://dx.doi.org/ /100004440wellcome trust 087622http://dx.doi.org/ /100004440wellcome trust104992issue-copyright-statement© the author 2017 